Temporal and spatial regulation of bone morphogenetic protein signaling in late lung development.

Bone morphogenetic proteins (BMPs) play important roles in early lung development. No study to date has addressed a role for BMP signaling in late lung development. We describe changes in the expression and localization of BMP receptors (Bmpr1a, Bmpr1b, and Bmpr2) and Smad (Smad1, Smad4, Smad5, and Smad8) intracellular signaling proteins during the saccular and alveolarization stages of late lung development. BMP signaling, assessed by Smad1/5 phosphorylation, nuclear translocation, and induction of id1, id2, and id3 gene expression, was evident throughout late lung development. Our data indicate that BMP signaling is active during late lung development, and points to roles for the BMP system in septal and vascular development, and in the homeostasis of the epithelial layer of large conducting airways in the mature lung.     

Enlarged meristems and delayed growth in plp mutants result from lack of CaaX prenyltransferases

Meristems require a myriad of intercellular signaling pathways for coordination of cell division within and between functional zones and clonal cell layers. This control of cell division ensures a constant availability of stem cells throughout the life span of the meristem while limiting overproliferation of meristematic cells and maintaining the meristem structure. We have undertaken a genetic screen to identify additional components of meristem signaling pathways. We identified pluripetala (plp) mutants based on their dramatically larger meristems and increased floral organ number. PLURIPETALA encodes the alpha-subunit shared between protein farnesyltransferase and protein geranylgeranyltransferase-I. plp mutants also have altered abscisic acid responses and overall much slower growth rate. plp is epistatic to mutations in the beta-subunit of farnesyltransferase and shows a synergistic interaction with clavata3 mutants. plp mutants lead to insights into the mechanism of meristem homeostasis and provide a unique in vivo system for studying the functional role of prenylation in eukaryotes.     

Involvement of a chloroplast HSP70 heat shock protein in the integration of a protein (light-harvesting complex protein precursor) into the thylakoid membrane

Molecular chaperones, including those belonging to the 70-kDa family of heat shock proteins (HSP70), assist both the translocation of proteins across membranes and their assembly into oligomeric complexes. We purified a chloroplast HSP70 (ct-HSP70) and demonstrated that it plays a major role in the insertion of the precursor of the major light-harvesting complex of photosystem II (pLHCP; an integral membrane protein) into the thylakoids (the inner membranes of the chloroplast). Addition of the purified ct-HSP70 is necessary for efficient insertion of pLHCP into isolated thylakoid membranes. This activity of the purified ct-HSP70 is similar to that previously reported for the total stromal extract. When the chloroplast stromal extract is depleted of HSP70, a correlative reduction in the insertion activity of pLHCP is observed. The interaction between the ct-HSP70 and pLHCP involves physical association. The purified HSP70 acts directly on the membrane protein, presumably prevents its refolding, and thereby helps to maintain its competence for insertion into membranes.     

Trypanosome-derived oligopeptidase B is released into the plasma of infected rodents, where it persists and retains full catalytic activity

A trypsin-like serine peptidase activity, levels of which correlate with blood parasitemia levels, is present in the plasma of rats acutely infected with Trypanosoma brucei brucei. Antibodies to a trypanosome peptidase with a trypsin-like substrate specificity (oligopeptidase B [OP-Tb]) cross-reacted with a protein in the plasma of trypanosome-infected rats on a Western blot. These antibodies also abolished 80% of the activity in the plasma of trypanosome-infected rats, suggesting that the activity may be attributable to a parasite-derived peptidase. We purified the enzyme responsible for the bulk of this activity from parasite-free T. b. brucei-infected rat plasma and confirmed its identity by protein sequencing. We show that live trypanosomes do not release OP-Tb in vitro and propose that disrupted parasites release it into the host circulation, where it is unregulated and retains full catalytic activity and may thus play a role in the pathogenesis of African trypanosomiasis.     

Alveolar fluid clearance in acute lung injury: what have we learned from animal models and clinical studies?

Background Acute lung injury and the acute respiratory distress syndrome continue to be significant causes of morbidity and mortality in the intensive care setting. The failure of patients to resolve the alveolar edema associated with these conditions is a major contributing factor to mortality; hence there is continued interest to understand the mechanisms of alveolar edema fluid clearance. Discussion The accompanying review by Vadász et al. details our current understanding of the signaling mechanisms and cellular processes that facilitate clearance of edema fluid from the alveolar compartment, and how these signaling processes may be exploited in the development of novel therapeutic strategies. To complement that report this review focuses on how intact organ and animal models and clinical studies have facilitated our understanding of alveolar edema fluid clearance in acute lung injury and acute respiratory distress syndrome. Furthermore, it considers how what we have learned from these animal and organ models and clinical studies has suggested novel therapeutic avenues to pursue. This article refers to the articles available at: and     

From the Cover: PNAS Plus: Chloride transport-driven alveolar fluid secretion is a major contributor to cardiogenic lung edema

Alveolar fluid clearance driven by active epithelial Na⁺ and secondary Cl⁻ absorption counteracts edema formation in the intact lung. Recently, we showed that impairment of alveolar fluid clearance because of inhibition of epithelial Na⁺ channels (ENaCs) promotes cardiogenic lung edema. Concomitantly, we observed a reversal of alveolar fluid clearance, suggesting that reversed transepithelial ion transport may promote lung edema by driving active alveolar fluid secretion. We, therefore, hypothesized that alveolar ion and fluid secretion may constitute a pathomechanism in lung edema and aimed to identify underlying molecular pathways. In isolated perfused lungs, alveolar fluid clearance and secretion were determined by a double-indicator dilution technique. Transepithelial Cl⁻ secretion and alveolar Cl⁻ influx were quantified by radionuclide tracing and alveolar Cl⁻ imaging, respectively. Elevated hydrostatic pressure induced ouabain-sensitive alveolar fluid secretion that coincided with transepithelial Cl⁻ secretion and alveolar Cl⁻ influx. Inhibition of either cystic fibrosis transmembrane conductance regulator (CFTR) or Na⁺-K⁺-Cl⁻ cotransporters (NKCC) blocked alveolar fluid secretion, and lungs of CFTR^−/− mice were protected from hydrostatic edema. Inhibition of ENaC by amiloride reproduced alveolar fluid and Cl⁻ secretion that were again CFTR-, NKCC-, and Na⁺-K⁺-ATPase–dependent. Our findings show a reversal of transepithelial Cl⁻ and fluid flux from absorptive to secretory mode at hydrostatic stress. Alveolar Cl⁻ and fluid secretion are triggered by ENaC inhibition and mediated by NKCC and CFTR. Our results characterize an innovative mechanism of cardiogenic edema formation and identify NKCC1 as a unique therapeutic target in cardiogenic lung edema.Traditionally, the formation of cardiogenic pulmonary edema has been attributed to passive fluid filtration across an intact alveolocapillary barrier along an increased hydrostatic pressure gradient. However, recent studies show that cardiogenic edema is critically regulated by active signaling processes. Activation of mechanosensitive endothelial ion channels increases lung vascular permeability (1), whereas alveolar epithelial cells lose their physiological ability to clear the distal airspaces from excess fluid by their capacity to actively transport ions across the epithelial barrier (2–4).In the intact lung, the predominant force driving alveolar fluid clearance is an active transepithelial Na⁺ transport from the alveolar into the interstitial space. A major portion of the apical Na⁺ entry is mediated by the amiloride-inhibitable epithelial Na⁺ channel (ENaC), with basolateral Na⁺ extrusion through the Na⁺-K⁺-ATPase (5). Cl⁻ and water are considered to follow paracellularly for electroneutrality and osmotic balance. In cardiogenic lung edema, the physiological protection against alveolar flooding provided by an intact alveolar fluid clearance is largely attenuated (3, 4). Previously, we have outlined the signaling events at the alveolocapillary barrier that underlie this inhibition of alveolar fluid clearance by showing that hydrostatic stress increases endothelial NO production in lung capillaries (6), which in turn, blocks alveolar Na⁺ and liquid absorption by a cGMP-dependent inhibition of epithelial ENaC (2).Unexpectedly, however, we observed that increased hydrostatic pressure not only blocks alveolar fluid clearance but reverses transepithelial fluid transport, resulting in effective alveolar fluid secretion that accounts for up to 70% of the total alveolar fluid influx at elevated hydrostatic pressure (2). This effect is not explicable by impaired alveolar fluid clearance and/or passive fluid leakage, and thus, it points to a previously unrecognized and potentially therapeutically exploitable pathomechanism in cardiogenic lung edema, namely alveolar fluid secretion driven by active transepithelial ion transport.Here, we aimed to analyze alveolar fluid secretion and its underlying cellular mechanisms in cardiogenic lung edema. We considered the Cl⁻ channel cystic fibrosis transmembrane conductance regulator (CFTR) as a putative key ion channel in this scenario, because it permits bidirectional permeation of anions under physiologically relevant conditions (7). Hence, the direction of Cl⁻ flux by CFTR may reverse depending on actual electrochemical gradients, thus turning an absorptive into a secretory epithelium or vice versa. This notion is supported by reports describing CFTR as both an absorptive and secretory channel in the regulation of alveolar fluid homeostasis (8, 9). By a combination of indicator dilution, imaging, and radioactive tracer techniques for the measurement of alveolar ion and fluid fluxes in the isolated lung, we show a critical role for CFTR-mediated Cl⁻ secretion in cardiogenic lung edema and identify the Na⁺-K⁺-2Cl⁻ cotransporter 1 (NKCC1) as a therapeutic target in this pathology.