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

OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis

Sixteen ovarian tumor (OTU) family deubiquitinases (DUBs) exist in humans, and most members regulate cell-signaling cascades. Several OTU DUBs were reported to be ubiquitin (Ub) chain linkage specific, but comprehensive analyses are missing, and the underlying mechanisms of linkage specificity are unclear. Using Ub chains of all eight linkage types, we reveal that most human OTU enzymes are linkage specific, preferring one, two, or a defined subset of linkage types, including unstudied atypical Ub chains. Biochemical analysis and five crystal structures of OTU DUBs with or without Ub substrates reveal four mechanisms of linkage specificity. Additional Ub-binding domains, the ubiquitinated sequence in the substrate, and defined S1’ and S2 Ub-binding sites on the OTU domain enable OTU DUBs to distinguish linkage types. We introduce Ub chain restriction analysis, in which OTU DUBs are used as restriction enzymes to reveal linkage type and the relative abundance of Ub chains on substrates

Site-specific incorporation of phosphotyrosine using an expanded genetic code.

Access to phosphoproteins with stoichiometric and site-specific phosphorylation status is key to understanding the role of protein phosphorylation. Here we report an efficient method to generate pure, active phosphotyrosine-containing proteins by genetically encoding a stable phosphotyrosine analog that is convertible to native phosphotyrosine. We demonstrate its general compatibility with proteins of various sizes, phosphotyrosine sites and functions, and reveal a possible role of tyrosine phosphorylation in negative regulation of ubiquitination

Solar radiative transfer simulations in Saharan dust plumes: particle shapes and 3-D effect

Radiative fields of three-dimensional inhomogeneous Saharan dust clouds have been calculated at solar wavelength (0.6 μm) by means of a Monte Carlo radiative transfer model. Scattering properties are taken from measurements in the SAMUM campaigns, from light scattering calculations for spheroids based on the MIESCHKA code, from Mie theory for spheres and from the geometric optics method assuming irregular shaped particles. Optical properties of different projected area equivalent shapes are compared. Large differences in optical properties are found especially in the phase functions. Results of radiative transfer calculations based on the Monte Carlo method are shown exemplarily for one dust cloud simulated by the cloud resolving atmospheric circulation model LM-MUSCAT-DES. Shape-induced differences in the radiation fluxes are pronounced, for example, the domain averaged normalized radiance is about 30% lower in the case of a dust plume consisting of spheroids or irregular particles compared to spheres. The effect of net horizontal photon transport (3-D effect) on the reflected radiance fields is only notable at the largest gradients in optical thickness. For example, the reflectance at low sun position differs locally about 15% when horizontal photon transport is accounted for. ‘Sharp edges' due to 1-D calculations are smoothed out in the 3-D case

A disordered region controls cBAF activity via condensation and partner recruitment

Intrinsically disordered regions (IDRs) represent a large percentage of overall nuclear protein content. The prevailing dogma is that IDRs engage in non-specific interactions because they are poorly constrained by evolutionary selection. Here, we demonstrate that condensate formation and heterotypic interactions are distinct and separable features of an IDR within the ARID1A/B subunits of the mSWI/SNF chromatin remodeler, cBAF, and establish distinct sequence grammars underlying each contribution. Condensation is driven by uniformly distributed tyrosine residues, and partner interactions are mediated by non-random blocks rich in alanine, glycine, and glutamine residues. These features concentrate a specific cBAF protein-protein interaction network and are essential for chromatin localization and activity. Importantly, human disease-associated perturbations in ARID1B IDR sequence grammars disrupt cBAF function in cells. Together, these data identify IDR contributions to chromatin remodeling and explain how phase separation provides a mechanism through which both genomic localization and functional partner recruitment are achieved

Lysine 27 Ubiquitination of the Mitochondrial Transport Protein Miro Is Dependent on Serine 65 of the Parkin Ubiquitin Ligase

Mitochondrial transport plays an important role in matching mitochondrial distribution to localised energy production and calcium buffering requirements. Here we demonstrate that Miro1, an outer mitochondrial membrane (OMM) protein crucial for the regulation of mitochondrial trafficking and distribution, is a substrate of the PINK1/Parkin mitochondrial quality control system in human dopaminergic neuroblastoma cells. Moreover Miro1 turnover on damaged mitochondria is altered in Parkinson's disease (PD) patient derived fibroblasts containing a pathogenic mutation in the PARK2 gene (encoding Parkin). By analysing the kinetics of Miro1 ubiquitination we further demonstrate that mitochondrial damage triggers rapid (within minutes) and persistent K27 type ubiquitination of Miro1 on the OMM, dependent on PINK1 and Parkin. Proteasomal degradation of Miro1 is then seen on a slower timescale, within 2-3 hours of the onset of ubiquitination. We find Miro ubiquitination in dopaminergic neuroblastoma cells is independent of Miro1 phosphorylation at serine 156 (S156), but is dependent on the recently identified serine S65 residue within Parkin that is phosphorylated by PINK1. Interestingly we find that Miro1 can stabilise phospho-mutant versions of Parkin on the OMM, suggesting that Miro is also part of a Parkin receptor complex. Moreover, we demonstrate that S65 in Parkin is critical for regulating Miro levels upon mitochondrial damage in rodent cortical neurons. Our results provide new insights into the ubiquitination-dependent regulation of the Miro-mediated mitochondrial transport machinery by PINK1/Parkin and also suggest that disruption of this regulation may be implicated in PD pathogenesis

A survey of transcutaneous blood gas monitoring among European neonatal intensive care units

BACKGROUND: PCO(2 )and PO(2 )are important monitoring parameters in neonatal intensive care units (NICU). Compared to conventional blood gas measurements that cause significant blood loss in preterms, transcutaneous (tc) measurements allow continuous, non-invasive monitoring of blood gas levels. The aim of the study was to survey the usage and opinions among German speaking NICUs concerning tc blood gas monitoring. METHODS: A questionnaire was developed and sent to 56 head nurses of different NICUs in Germany, Switzerland and Austria. RESULTS: A completely answered questionnaire was obtained from 41 NICUs. In two of these units tc measurements are not performed. In most NICUs (77%), both P(tc)O(2 )and P(tc)CO(2 )are measured simultaneously. Most units change the sensors every 3 hours; however, the recommended temperature of 44°C is used in only 15% of units. In only 8% of units are arterial blood gases obtained to validate tc values. Large variations were found concerning the targeted level of oxygen saturation [median upper limit: 95% (range 80–100%); median lower limit: 86% (range 75–93%)] and PO(2 )[median upper limit: 70 mmHg (range 45–90 mmHg); median lower limit: 44 mmHg (range 30–60 mmHg)]. CONCLUSION: Our survey shows that the use of tc monitors remains widespread among German speaking NICUs, despite earlier data suggesting that their use had been abandoned in many NICUs worldwide. In addition, we suggest that the current method of monitoring oxygenation may not prevent hyperoxemia in preterm infants

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

A Realistic Validation Study of a New Nitrogen Multiple-Breath Washout System

Background For reliable assessment of ventilation inhomogeneity, multiple-breath washout (MBW) systems should be realistically validated. We describe a new lung model for in vitro validation under physiological conditions and the assessment of a new nitrogen (N2)MBW system. Methods The N2MBW setup indirectly measures the N2 fraction (FN2) from main-stream carbon dioxide (CO2) and side-stream oxygen (O2) signals: FN2 = 1−FO2−FCO2−FArgon. For in vitro N2MBW, a double chamber plastic lung model was filled with water, heated to 37°C, and ventilated at various lung volumes, respiratory rates, and FCO2. In vivo N2MBW was undertaken in triplets on two occasions in 30 healthy adults. Primary N2MBW outcome was functional residual capacity (FRC). We assessed in vitro error (√[difference]2) between measured and model FRC (100–4174 mL), and error between tests of in vivo FRC, lung clearance index (LCI), and normalized phase III slope indices (Sacin and Scond). Results The model generated 145 FRCs under BTPS conditions and various breathing patterns. Mean (SD) error was 2.3 (1.7)%. In 500 to 4174 mL FRCs, 121 (98%) of FRCs were within 5%. In 100 to 400 mL FRCs, the error was better than 7%. In vivo FRC error between tests was 10.1 (8.2)%. LCI was the most reproducible ventilation inhomogeneity index. Conclusion The lung model generates lung volumes under the conditions encountered during clinical MBW testing and enables realistic validation of MBW systems. The new N2MBW system reliably measures lung volumes and delivers reproducible LCI values

Pollination and Predation Limit Fruit Set in a Shrub, Bourreria succulents (Boraginaceae), after Hurricanes on San Salvador Island, Bahamas 1

Hurricanes have been assumed to reduce the reproduction of plants, either directly by leaf stripping and stress or indirectly by reducing pollinators. I examined the pollination and fruit set of a common shrub, Bourreria succulenta , after hurricanes on San Salvador island, Bahamas. Contrary to the assumption of resource limitation, B. succulenta showed unusually prolific flowering after Hurricane Lili stripped leaves from most of the plants in October 1996. I predicted that the abundant flowering would saturate pollinators and that fruit set would be pollination-limited. Fruit set was strongly pollination-limited by 71 percent. Butterflies are probably the major pollinators and were present at the site, but they rarely visited B. succulenta flowers even though flowers were brimming with nectar. Nectarivorous birds (Bananaquits and Bahama Wbodstars) visit B. succulenta flowers, but their populations were decimated by Hurricane Lili and they rarely visited flowers during this time. Fruit set was also severely predation-limited; a moth caterpillar (Gelechiidae) was extremely abundant and ate buds, flowers, and fruits, causing a further 68 percent reduction in fruit set. Together, pollination limitation and predation limitation reduced fruit set to only 7 percent or less. Predation was also intense in 1999 after Hurricane Floyd and resulted in 11 percent fruit set or less. Whether or not hurricanes were the cause of limited pollinators or abundant predators, the resulting low fruit set could have population effects because hurricanes can provide opportunities for the recruitment of new plants. These results emphasize that understanding plant–animal interactions may be necessary for predicting the effects of hurricanes on plant reproductive success, which may affect subsequent recruitment. Species on small islands like San Salvador (150 km 2 ) with relatively few species may be especially vulnerable to environmental disturbances such as hurricanes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75721/1/j.1744-7429.2001.tb00184.x.pd