Mechanism of endothelial cell shape change in oxidant injury

Changes in endothelial cell morphology induced by neutrophil-generated hydrogen peroxide (H2O2) may account for the capillary leak of the adult respiratory distress syndrome (ARDS). The relationship of H2O2 effects on the concentration of intracellular Ca2+([Ca2+]i) and ATP to changes in microfilaments and microtubules, important determinants of cell shape, was examined. Bovine pulmonary artery endothelial cells were injured over a 2-hr time course with a range of H2O2 doses (0-20 mM). The higher concentrations of H2O2 consistently produced contraction and rounding of>50-75% of cells by 1-2 hr. The range of 1-20 mM H2O2 produced rapid, significant reductions in endothelial ATP levels over the time course of injury. Although there were significant increases in mean endothelial [Ca2+]i in response to 5, 10, and 20 mM H2O2, 1 mMH2O2 did not affect the [Ca2+]i. Fluorescence microscopy revealed that microfilament disruption occurred as ATP levels fell and preceded depolymerization of microtubules which developed after [Ca2+]1 approached 1 x 10-6 M. H2O2 at 1 mM injury caused microfilament disruption but did not depolymerize microtubules. Microfilament disruption occurred without oxidant exposure, when ATP levels were reduced by glucose depletion and mitochondrial inhibition with oligomycin (650 nM). If a Ca2+ ionophore, ionomycin (5 [mu]M), was then added, [Ca2+]i rose to > 1 x 10-6 M, microtubules fragmented and depolymerized, and cell contraction and rounding very similar to that induced by H2O2 occurred. These results suggest that endothelial cell dysfunction and capillary leak in ARDS may be due to H2O2-mediated changes in cellular ATP and [Ca2+]i.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27994/1/0000428.pd

Actin polymerization in cellular oxidant injury

Microfilaments undergo an ATP-dependent disruption into shortened bundles following cellular exposure to oxidants. This phenomenon does not require a net change in the amount of polymerized actin. However, increased amounts of polymerized actin have been detected in oxidant-injured cells and it was the purpose of this study to determine the conditions under which the actin polymerization may occur. Utilizing the formation of oxidized glutathione (GSSG) as an indicator of cellular sulfhydryl oxidation, conditions were chosen to accentuate sulfhydryl oxidation within the target P388D1 cell line following exposure to the oxidants, H2O2 and diamide. Using the DNase I and flow cytometric assays of actin polymerization, significant polymerization of actin was detected only under conditions in which sulfhydryl oxidation occurred after exposure to the two oxidizing agents. Greater sulfhydryl oxidation early in the course of injury was associated with a greater rate and extent of actin polymerization in the injured cells. Experiments with cells depleted of glutathione (GSH) demonstrated that neither loss of GSH nor absolute levels of GSSG formed during oxidant exposure were responsible for the polymerization of actin. The data presented are consistent with the hypothesis that oxidizing conditions which induce significant sulfhydryl oxidation in target cells are correlated with assembly of polymerized actin and that this represents a process which is distinct and separate from the ATP-dependent gross disruption of microfilaments.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29200/1/0000254.pd

Survey of overnight academic hospitalist supervision of trainees

In 2003, Accreditation Council for Graduate Medical Education (ACGME) announced the first in a series of guidelines related to the residency training. The most recent recommendations include explicit recommendations regarding the provision of on‐site clinical supervision for trainees of internal medicine. To meet these standards, many internal medicine residency programs turned to hospitalist programs to fill that need. However, much is unknown about the current relationships between hospitalist and residency programs, specifically with regard to supervisory roles and supervision policies. We aimed to describe how academic hospitalists currently supervise housestaff during the on‐call, or overnight, period and hospitalist program leader their perceptions of how these new policies would impact trainee‐hospitalist interactions. Journal of Hospital Medicine 2012; © 2012 Society of Hospital MedicinePeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/93713/1/1961_ftp.pd

EEF2 Analysis Challenges the Monophyly of Archaeplastida and Chromalveolata

BACKGROUND: Classification of eukaryotes provides a fundamental phylogenetic framework for ecological, medical, and industrial research. In recent years eukaryotes have been classified into six major supergroups: Amoebozoa, Archaeplastida, Chromalveolata, Excavata, Opisthokonta, and Rhizaria. According to this supergroup classification, Archaeplastida and Chromalveolata each arose from a single plastid-generating endosymbiotic event involving a cyanobacterium (Archaeplastida) or red alga (Chromalveolata). Although the plastids within members of the Archaeplastida and Chromalveolata share some features, no nucleocytoplasmic synapomorphies supporting these supergroups are currently known. METHODOLOGY/PRINCIPAL FINDINGS: This study was designed to test the validity of the Archaeplastida and Chromalveolata through the analysis of nucleus-encoded eukaryotic translation elongation factor 2 (EEF2) and cytosolic heat-shock protein of 70 kDa (HSP70) sequences generated from the glaucophyte Cyanophora paradoxa, the cryptophytes Goniomonas truncata and Guillardia theta, the katablepharid Leucocryptos marina, the rhizarian Thaumatomonas sp. and the green alga Mesostigma viride. The HSP70 phylogeny was largely unresolved except for certain well-established groups. In contrast, EEF2 phylogeny recovered many well-established eukaryotic groups and, most interestingly, revealed a well-supported clade composed of cryptophytes, katablepharids, haptophytes, rhodophytes, and Viridiplantae (green algae and land plants). This clade is further supported by the presence of a two amino acid signature within EEF2, which appears to have arisen from amino acid replacement before the common origin of these eukaryotic groups. CONCLUSIONS/SIGNIFICANCE: Our EEF2 analysis strongly refutes the monophyly of the Archaeplastida and the Chromalveolata, adding to a growing body of evidence that limits the utility of these supergroups. In view of EEF2 phylogeny and other morphological evidence, we discuss the possibility of an alternative eukaryotic supergroup

Decreased mitochondrial respiration in aneurysmal aortas of Fibulin-4 mutant mice is linked to PGC1A regulation

Aim Thoracic aortic aneurysms are a life-threatening condition often diagnosed too late. To discover novel robust biomarkers, we aimed to better understand the molecular mechanisms underlying aneurysm formation. Methods and results In Fibulin-4R/R mice, the extracellular matrix protein Fibulin-4 is 4-fold reduced, resulting in progressive ascending aneurysm formation and early death around 3 months of age. We performed proteomics and genomics studies on Fibulin-4R/R mouse aortas. Intriguingly, we observed alterations in mitochondrial protein composition in Fibulin-4R/R aortas. Consistently, functional studies in Fibulin-4R/R vascular smooth muscle cells (VSMCs) revealed lower oxygen consumption rates, but increased acidification rates. Yet, mitochondria in Fibulin-4R/R VSMCs showed no aberrant cytoplasmic localization. We found similar reduced mitochondrial respiration in Tgfbr-1M318R/+ VSMCs, a mouse model for Loeys-Dietz syndrome (LDS). Interestingly, also human fibroblasts from Marfan (FBN1) and LDS (TGFBR2 and SMAD3) patients showed lower oxygen consumption. While individual mitochondrial Complexes I–V activities were unaltered in Fibulin-4R/R heart and muscle, these tissues showed similar decreased oxygen consumption. Furthermore, aortas of aneurysmal Fibulin-4R/R mice displayed increased reactive oxygen species (ROS) levels. Consistent with these findings, gene expression analyses revealed dysregulation of metabolic pathways. Accordingly, blood ketone levels of Fibulin-4R/R mice were reduced and liver fatty acids were decreased, while liver glycogen was increased, indicating dysregulated metabolism at the organismal level. As predicted by gene expression analysis, the activity of PGC1α, a key regulator between mitochondrial function and organismal metabolism, was downregulated in Fibulin-4R/R VSMCs. Increased TGFβ reduced PGC1α levels, indicating involvement of TGFβ signalling in PGC1α regulation. Activation of PGC1α restored the decreased oxygen consumption in Fibulin-4R/R VSMCs and improved their reduced growth potential, emphasizing the importance of this key regulator. Conclusion Our data indicate altered mitochondrial function and metabolic dysregulation, leading to increased ROS levels and altered energy production, as a novel mechanism, which may contribute to thoracic aortic aneurysm formation

Protective effect of glutamine on endothelial cell ATP in oxidant injury

Endothelial cell dysfunction following exposure to H2O2 is associated with rapid inhibition of glucose-dependent pathways of ATP synthesis. The role other substrates for ATP synthesis (e.g., amino acids) may play in the metabolism of H2O2-injured cells is unclear. The effect of glutamine, a precursor of the Kreb's cycle intermediate [alpha]-ketoglutarate on ATP levels in bovine pulmonary artery endothelial cells exposed to H2O2 was examined. The presence of glutamine during H2O2 injury significantly enhanced ATP levels in the injured cells. Concentrations of glutamine as low as 50 [mu]M produced significant improvement of ATP levels in endothelial cells exposed to 5 mM H2O2. The 2 mM concentration of glutamine produced the greatest benefit, while greater concentrations of glutamine (5-20 mM) were actually associated with progressive decrements of the maximal benefit seen with the 2 mM concentration. The 2 mM concentration of glutamine produced similar enhancement of ATP with 1 and 10 mM H2O2 injury as well. Short-term viability following 5 mM H2O2 injury was significantly improved by the presence of 2 mM glutamine. The most effective concentration of glutamine (2 mM) did not scavenge H2O2 in a fluorometric assay. These observations suggest that mitochondrial substrates, such as glutamine, that bypass glucose-dependent pathways of ATP synthesis may be useful therapeutic agents for maintenance of ATP levels in oxidant-injured cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28408/1/0000183.pd