Alterations of alveolar type II cells and intraalveolar surfactant after bronchoalveolar lavage and perfluorocarbon ventilation. An electron microscopical and stereological study in the rat lung

<p>Abstract</p> <p>Background</p> <p>Repeated bronchoalveolar lavage (BAL) has been used in animals to induce surfactant depletion and to study therapeutical interventions of subsequent respiratory insufficiency. Intratracheal administration of surface active agents such as perfluorocarbons (PFC) can prevent the alveolar collapse in surfactant depleted lungs. However, it is not known how BAL or subsequent PFC administration affect the intracellular and intraalveolar surfactant pool.</p> <p>Methods</p> <p>Male wistar rats were surfactant depleted by BAL and treated for 1 hour by conventional mechanical ventilation (<it>Lavaged-Gas</it>, n = 5) or partial liquid ventilation with PF 5080 (<it>Lavaged-PF5080</it>, n = 5). For control, 10 healthy animals with gas (<it>Healthy-Gas</it>, n = 5) or PF5080 filled lungs (<it>Healthy-PF5080</it>, n = 5) were studied. A design-based stereological approach was used for quantification of lung parenchyma and the intracellular and intraalveolar surfactant pool at the light and electron microscopic level.</p> <p>Results</p> <p>Compared to <it>Healthy</it>-lungs, <it>Lavaged</it>-animals had more type II cells with lamellar bodies in the process of secretion and freshly secreted lamellar body-like surfactant forms in the alveoli. The fraction of alveolar epithelial surface area covered with surfactant and total intraalveolar surfactant content were significantly smaller in <it>Lavaged</it>-animals. Compared with <it>Gas</it>-filled lungs, both <it>PF5080</it>-groups had a significantly higher total lung volume, but no other differences.</p> <p>Conclusion</p> <p>After BAL-induced alveolar surfactant depletion the amount of intracellularly stored surfactant is about half as high as in healthy animals. In lavaged animals short time liquid ventilation with PF5080 did not alter intra- or extracellular surfactant content or subtype composition.</p

Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Serine⁶⁵

We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser(65) within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser(65) that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser(65) in ubiquitin lies in a similar motif to Ser(65) in the Ubl domain of Parkin. Remarkably, PlNK1 directly phosphorylates Ser(65) of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser(65) (Delta Ubl-Parkin) is robustly activated by ubiquitin(Phospho-Ser65), but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser(65) (Ubl(phospho-Ser65)) can also activate Delta Ubl-Parkin similarly to ubiquitin(PhosPh-Ser65). Thirdly, we establish that ubiquitin(PhosPh-Ser65), but not non-phosphorylated ubiquitin or Ubl(PhosPh-Ser65) activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser(65) and ubiquitin at Ser(65), since only mutation of both proteins at Ser(65) completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser(65), but also by phosphorylating ubiquitin at Ser(65). We propose that phosphorylation of Parkin at Ser(65) serves to prime the E3 ligase enzyme for activation by ubiquitin(PhosPh-Ser65), suggesting that small molecules that mimic ubiquitin(PhosPh-Ser65) could hold promise as novel therapies for Parkinson's disease

Alterations of alveolar type II cells and intraalveolar surfactant after bronchoalveolar lavage and perfluorocarbon ventilation. An electron microscopical and stereological study in the rat lung-2

<p><b>Copyright information:</b></p><p>Taken from "Alterations of alveolar type II cells and intraalveolar surfactant after bronchoalveolar lavage and perfluorocarbon ventilation. An electron microscopical and stereological study in the rat lung"</p><p>http://respiratory-research.com/content/8/1/40</p><p>Respiratory Research 2007;8(1):40-40.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892019.</p><p></p>ular myelin with its characteristic lattice-like structure in the healthy animals is exemplarly shown in (A). Multi- and unilamellar surfactant forms are shown in (B). Tubular myelin was only extremely rarely seen in the lavaged animals where multi- and unilamellar forms (C) and numerous lamellar body-like forms (D) were present

Alterations of alveolar type II cells and intraalveolar surfactant after bronchoalveolar lavage and perfluorocarbon ventilation. An electron microscopical and stereological study in the rat lung-4

<p><b>Copyright information:</b></p><p>Taken from "Alterations of alveolar type II cells and intraalveolar surfactant after bronchoalveolar lavage and perfluorocarbon ventilation. An electron microscopical and stereological study in the rat lung"</p><p>http://respiratory-research.com/content/8/1/40</p><p>Respiratory Research 2007;8(1):40-40.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892019.</p><p></p>iment. Values in the PF5080 group are significantly higher than in gas ventilated animals (* p < 0.0001)

Alterations of alveolar type II cells and intraalveolar surfactant after bronchoalveolar lavage and perfluorocarbon ventilation. An electron microscopical and stereological study in the rat lung-3

<p><b>Copyright information:</b></p><p>Taken from "Alterations of alveolar type II cells and intraalveolar surfactant after bronchoalveolar lavage and perfluorocarbon ventilation. An electron microscopical and stereological study in the rat lung"</p><p>http://respiratory-research.com/content/8/1/40</p><p>Respiratory Research 2007;8(1):40-40.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892019.</p><p></p>ile all four different intraalveolar surfactant subtypes (lamellar body-like forms = lbl, tubular myelin = tm, multilamellar vesicles = mv, unilamellar vesicles = uv) were present in healthy animals, there were no measurable amounts of tubular myelin and decreased fractions of unilamellar vesicles in lavaged animals. This was counterbalanced by increased fractions of lamellar body-like forms and multilamellar vesicles in the lavaged groups, indicating a relative increase in freshly secreted surfactant material in the alveoli

Alterations of alveolar type II cells and intraalveolar surfactant after bronchoalveolar lavage and perfluorocarbon ventilation. An electron microscopical and stereological study in the rat lung-1

<p><b>Copyright information:</b></p><p>Taken from "Alterations of alveolar type II cells and intraalveolar surfactant after bronchoalveolar lavage and perfluorocarbon ventilation. An electron microscopical and stereological study in the rat lung"</p><p>http://respiratory-research.com/content/8/1/40</p><p>Respiratory Research 2007;8(1):40-40.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892019.</p><p></p>ies appear normal in number in the Healthy-Gas (A) and Healthy-PF5080 (B) groups, while the type II cells seem to be smaller in size and contain less lamellar bodies in the Lavaged-Gas (C) and Lavaged-PF5080 (D) groups. Lamellar bodies in the process of secretion were seen more frequently in the lavaged animals (exemplarly shown in D)