Combined results from laser capture microdissection of mouse airway epithelial cells followed by high power (MALDI-FTICR) MS, and fluorescent two-dimensional gel elctrophoresis (2D-DIGE) of the whole lung, allowed us to identify proteins differentially expressed after naphthalene induced airway injury. Further, we discovered several novel aspects of Cystic Fibrosis (CF) lung pathology in an F508del-Cftr mouse model using this approach. The combined MALDI-FTICR-MS and 2D-DIGE data show that lung carbonyl reductase (CBR2), involved in prostaglandin metabolism, converting PGE2 to PGF2alpha, is localized to airway cells and is reduced 2-fold in mutant mice compared to normal, both before and after challenge. Further, we observe a downregulation of two key enzymes of retinoic acid metabolism after injury, which is more pronounced in CF mutant mice. These data show that state-of-the-art proteomics can be used to evaluate airway injury in small cell samples. Further, the results suggest the involvement of prostaglandin and retinoic acid metabolism in the abnormal responses of CF mutant mice to injury.
"Global comparative analyses of CF versus non-CF samples have been used to identify differentially expressed proteins in human bronchoalveolar lavage fluid (BALF) [20,21], sputum , bronchial biopsy tissue , serum  and cultured epithelial cells [25,26], and in mouse lung and colonic tissue [27-29]. Many of the proteins highlighted by global analyses can be related functionally to biological processes and pathways known to contribute to CF disease pathogenesis, including chronic inflammation, proteolytic activity and oxidative stress response proteins. "
[Show abstract][Hide abstract] ABSTRACT: Cystic fibrosis is one of the most common life-limiting inherited disorders. Its clinical impact manifests chiefly in the lung, pancreas, gastrointestinal tract and sweat glands, with lung disease typically being most detrimental to health. The median age for survival has increased dramatically over the past decades, largely thanks to advances in understanding of the mechanisms and consequences of disease, leading to the development of better therapies and treatment regimes. The discovery of dysregulated protein biomarkers linked to cystic fibrosis has contributed considerably to this end. This article outlines clinical trials targeting known protein biomarkers, and the current and future contributions of proteomic techniques to cystic fibrosis research. The treatments described range from those designed to provide functional copies of the mutant protein responsible for cystic fibrosis, to others addressing the associated symptoms of chronic inflammation. Preclinical research has employed proteomics to help elucidate pathways and processes implicated in disease that might present opportunities for therapy or prognosis. Global analyses of cystic fibrosis have detected the differential expression of proteins involved in inflammation, proteolytic activity and oxidative stress, which are recognized symptoms of the cystic fibrosis phenotype. The dysregulation of other processes, such as the complement and mitochondrial systems, has also been implicated. A number of studies have focused specifically on proteins that interact with the cystic fibrosis protein, with the goal of restoring its normal proteostasis. Consequently, proteins involved in synthesis, folding, degradation, translocation and localization of the protein have been identified as potential therapeutic targets. Cystic fibrosis patients are prone to lung infections that are thought to contribute to chronic inflammation, and thus proteomic studies have also searched for microbiological biomarkers to use in early infection diagnosis or as indicators of virulence. The review concludes by proposing a future role for proteomics in the high-throughput validation of protein biomarkers under consideration as outcome measures for use in clinical trials and routine disease monitoring.
Genome Medicine 12/2010; 2(12):88. DOI:10.1186/gm209 · 5.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in humans is frequently associated with progressive liver disease, which appears to result from obstruction of biliary ducts with mucous material. CFTR in the liver is expressed in the biliary epithelium. With the use of a mouse model for cystic fibrosis (CF) we have studied the relationship between CFTR expression and glycoprotein secretion in primary culture of mouse gallbladder epithelial cells (MGBC) MGBC in culture maintain a well-differentiated phenotype as shown by microscopy. The cells produce CFTR mRNA to levels comparable to the intact tissue. With patch-clamp analysis we could frequently observe a linear protein kinase A-regulated Cl- channel that shows all the major characteristics of human CFTR, although its conductance is lower (5 pS compared with 8 pS). MGBC in culture produce and secrete high molecular weight glycoproteins (HMG) in a time-dependent and temperature-sensitive manner. Secretion of HMG was not stimulated significantly by either adenosine 3',5'-cyclic monophosphate (cAMP), Ca2+, or protein kinase C agonists in this system. High concentrations (3 mM) of extracellular ATP stimulated secretion threefold, but low concentrations (0.3 mM) had no effect. Approximately one-third of the HMG produced and secreted consisted of mucin. Cultured MGBC from CFTR-deficient mice produced and secreted mucin to a similar extent as normal cells. We conclude that cultured mouse gallbladder cells are a convenient model to study both CFTR function and mucin secretion. In this system, we found no evidence for a direct link between mucin secretion and CFTR activity, as has been suggested for other cell types.
The American journal of physiology 01/1997; 271(6 Pt 1):G1074-83. · 3.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The major principal direction alignment (MPDA) principle, developed in the context of robust control theory, states that a necessary and sufficient condition for the spectral radius of a matrix to equal its maximum singular value is that the major input and the major output principal directions of the matrix be aligned. The MPDA principle emerged from a study of the derivatives of the maximum singular value. An ambiguity that occurs when the maximum singular value is repeated is considered in this paper, together with a modified statement of the major principal direction alignment principle. The new necessary and sufficient condition for the spectral radius of a matrix to equal its maximum singular value is that there exists at least one major input and output principal direction pair of the matrix that is aligned. A rigorous proof is provided for the new necessary and sufficient condition, which makes use of early results on dual norms and dual vectors. An example is presented to illustrate the results.
American Control Conference, 2003. Proceedings of the 2003; 07/2003
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