Proteomic analysis of lamellar bodies isolated from rat lungs

<p>Abstract</p> <p>Background</p> <p>Lamellar bodies are lysosome-related secretory granules and store lung surfactant in alveolar type II cells. To better understand the mechanisms of surfactant secretion, we carried out proteomic analyses of lamellar bodies isolated from rat lungs.</p> <p>Results</p> <p>With peptide mass fingerprinting by Matrix Assisted Laser Desorption/Ionization – Time of Flight mass spectrometry, 44 proteins were identified with high confidence. These proteins fell into diverse functional categories: surfactant-related, membrane trafficking, calcium binding, signal transduction, cell structure, ion channels, protein processing and miscellaneous. Selected proteins were verified by Western blot and immunohistochemistry.</p> <p>Conclusion</p> <p>This proteomic profiling of lamellar bodies provides a basis for further investigations of functional roles of the identified proteins in lamellar body biogenesis and surfactant secretion.</p

Vacuolar ATPase Regulates Surfactant Secretion in Rat Alveolar Type II Cells by Modulating Lamellar Body Calcium

Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase) is the enzyme responsible for pumping H+ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase) dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1), an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca2+ chelator, BAPTA-AM, the protein kinase C (PKC) inhibitor, staurosporine, and the Ca2+/calmodulin-dependent protein kinase II (CaMKII), KN-62. Baf A1 induced Ca2+ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca2+ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca2+ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion

Characterization of Immunodominant and Potentially Protective Epitopes of Mannheimia haemolytica Serotype 1 Outer Membrane Lipoprotein PlpE

Mannheimia haemolytica serotype 1 (S1) is the most common bacterial isolate found in shipping fever pneumonia in beef cattle. Currently used vaccines against M. haemolytica do not provide complete protection against the disease. Research with M. haemolytica outer membrane proteins (OMPs) has shown that antibodies to one particular OMP from S1, PlpE, may be important in immunity. In a recently published work, members of our laboratory showed that recombinant PlpE (rPlpE) is highly immunogenic when injected subcutaneously into cattle and that the acquired immunity markedly enhanced resistance to experimental challenge (A. W. Confer, S. Ayalew, R. J. Panciera, M. Montelongo, L. C. Whitworth, and J. D. Hammer, Vaccine 21:2821-2829, 2003). The objective of this work was to identify epitopes of PlpE that are responsible for inducing the immune response. Western blot analysis of a series of rPlpE with nested deletions on both termini with bovine anti-PlpE hyperimmune sera showed that the immunodominant region is located close to the N terminus of PlpE. Fine epitope mapping, in which an array of overlapping 13-mer synthetic peptides attached to a derivatized cellulose membrane was probed with various affinity-purified anti-PlpE antibodies, identified eight highly reactive regions, of which region 2 (R2) was identified as the specific epitope. The R2 region is comprised of eight imperfect repeats of a hexapeptide (QAQNAP) and is located between residues 26 and 76. Complement-mediated bactericidal activity of affinity-purified anti-PlpE bovine antibodies confirmed that antibodies directed against the R2 region are effective in killing M. haemolytica

Hydrological responses to land use and land cover change and climate dynamics in the Rift Valley Lakes Basin, Ethiopia

Due to the rapid socio-economic development in the Ethiopian Rift Valley basin, the pressures on water resources are increasing. To understand the change of spatio-temporal water fluxes, the hydrologic model SWAT+ (Soil and Water Assessment Tool+) was applied to five selected watersheds within the basin. With regards to the objective functions, Kling–Gupta efficiency (KGE: 0.68–0.84), the Nash–Sutcliffe efficiency (NSE: 0.61–0.73), percent bias (PBIAS: −3.4 to 1.4), and RMSE-observations standard deviation ratio (RSR: 0.52–0.69), the SWAT+ model performed very well for daily streamflow in all watersheds. The change in water balance components indicated a considerable spatial variation of water fluxes in the watersheds. Precipitation, evapotranspiration, and infiltration have generally decreased, but surface runoff has increased in the interference period compared to the baseline period. The spatial distribution of rainfall (−40 to 10%), evapotranspiration (−20 to 5%), surface runoff (7.8–13.1%), lateral flow (4.47 to −16.5%), and percolation (−3.3 to −10.2%) varied. The changes in the hydrologic system within the basin are greatly attributed to the combination of land use and land cover change due to rapid population growth and climate variability. HIGHLIGHTS Examines the combined and isolated impacts of LULC change and climate dynamics on hydrological processes.; LULC change associated with rapid population growth and climate variability are significant drivers of changes in the hydrologic system.; The methodology employed in the study can serve as a valuable model for similar studies in other regions, and it contributes to the existing literature on hydrological responses.

Association of V-ATPase subunits with lipid rafts.

<p>Type II cells were lysed in 1% Triton X-100 (control) or 0.5% Triton X-100+0.5% saponin (cholesterol depleted). Later, the lysates were subjected to raft isolation and various fractions were collected. Equal volumes of fractions were immunoblotted for V-ATPase B1/2 and a1 subunits.</p

Schematic representation of proposed events for V-ATPase-mediated lung surfactant secretion.

<p>Following V-ATPase inhibition by Baf A1 or lung surfactant secretagogues, Ca²⁺ is mobilized from lamellar bodies. The small localized changes in Ca²⁺ concentration leads to the further release of Ca²⁺ from ER store. The global increase in intracellular Ca²⁺ concentration results in the activation of PKC and CaMKII and the increase in surfactant secretion. LB: lamellar bodies; ER: endoplasmic reticulum.</p

Subcellular localization of V-ATPase subunits.

<p>Total proteins isolated from plasma membrane (PM, 20 µg), lamellar bodies (LB, 5 µg), type II cells (T2, 20 µg), and lung tissue homogenates (LH, 20 µg) were immunoblotted for V-ATPase B1/2 and a1 subunits.</p