Hugh O'Brodovich

Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer.

Inefficient nuclear delivery of plasmid DNA is thought to be one of the daunting hurdles to gene transfer, utilizing a nonviral delivery system such as polycation–DNA complex. Following its internalization by endocytosis, plasmid DNA has to be released into the cytosol before its nuclear entry can occur. However, the stability of plasmid DNA in the cytoplasm, that may play a determinant role in the transfection efficiency, is not known. The turnover of plasmid DNA, delivered by microinjection into the cytosol, was determined by fluorescence in situ hybridization (FISH) and quantitative single-cell fluorescence video-image analysis. Both single- and double-stranded circular plasmid DNA disappeared with an apparent half-life of 50–90 min from the cytoplasm of HeLa and COS cells, while the amount of co-injected dextran (MW 70000) remained unaltered. We propose that cytosolic nuclease(s) are responsible for the rapid degradation of plasmid DNA, since (1) elimination of plasmid DNA cannot be attributed to cell division or to the activity of apoptotic and lysosomal nucleases; (2) disposal of microinjected plasmid DNA was inhibited in cytosol-depleted cells or following the encapsulation of DNA in phospholipid vesicles; (3) generation and subsequent elimination of free 3′-OH ends could be detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay (TUNEL), reflecting the fragmentation of the injected DNA; and finally (4) isolated cytosol, obtained by selective permeabilization of the plasma membrane, exhibits divalent cation-dependent, thermolabile nuclease activity, determined by Southern blotting and 32P-release from end-labeled DNA. Collectively, these findings suggest that the metabolic instability of plasmid DNA, caused by cytosolic nuclease, may constitute a previously unrecognized impediment for DNA translocation into the nucleus and a possible target to enhance the efficiency of gene delivery.

An SH3 binding region in the epithelial Na+ channel (alpha rENaC) mediates its localization at the apical membrane.

The amiloride‐sensitive Na+ channel constitutes the rate‐limiting step for Na+ transport in epithelia. Immunolocalization and electrophysiological studies have demonstrated that this channel is localized at the apical membrane of polarized epithelial cells. This localization is essential for proper channel function in Na+ transporting epithelia. In addition, the channel has been shown to associate with the cytoskeletal proteins ankyrin and alpha‐spectrin in renal epithelia. However, the molecular mechanisms underlying the cytoskeletal interactions and apical membrane localization of this channel are largely unknown. In this study we show that the putative pore forming subunit of the rat epithelial (amiloride‐sensitive) Na+ channel (alpha ENaC) binds to alpha‐spectrin in vivo, as determined by co‐immunoprecipitation. This binding is mediated by the SH3 domain of alpha‐spectrin which binds to a unique proline‐rich sequence within the C‐terminal region of alpha rENaC. Accordingly, the C‐terminal region is sufficient to mediate binding to intact alpha‐spectrin from alveolar epithelial cell lysate. When microinjected into the cytoplasm of polarized primary rat alveolar epithelial cells, a recombinant fusion protein containing the C‐terminal proline‐rich region of alpha rENaC localized exclusively to the apical area of the plasma membrane, as determined by confocal microscopy. This localization paralleled that of alpha‐spectrin. In contrast, microinjected fusion protein containing the N‐terminal (control) protein of alpha rENaC remained diffuse within the cytoplasm. These results suggest that an SH3 binding region in alpha rENaC mediates the apical localization of the Na+ channel. Thus, cytoskeletal interactions via SH3 domains may provide a novel mechanism for retaining proteins in specific membranes of polarized epithelial cells.

Efficacy of albuterol administered by nebulizer versus spacer device in children with acute asthma

The aim of this study was to compare the response to inhaled albuterol after administration by nebulizer with the response after administration by a metered-dose inhaler and spacer device (MDI-spacer) to children with acute asthma. In a double-blind fashion, 33 children (6 to 14 years of age) with forced expiratory volume in 1 second (FEV1) between 20% and 70% of predicted values, and who were seen in the emergency department with acute asthma, were studied. They were treated with aerosolized albuterol or placebo by MDI-spacer, followed immediately by albuterol or placebo administered by nebulizer with oxygen. The dose ratio for albuterol by MDI-spacer versus nebulizer was 1:5. Outcome measures included a clinical score, respiratory rate, arterial oxygen saturation, and FEV1, measured before and 10, 20, and 40 minutes after treatment. With the exception of heart rate (which increased in the nebulizer group and decreased in the MDI-spacer group (p < 0.05), no difference in the rate of improvement of clinical score, respiratory rate, arterial oxygen saturation, or FEV1 was noted during the 40-minute study period between children who received albuterol by nebulizer and those who received it by MDI-spacer. We conclude that spacers and nebulizers are equally effective means of delivering beta 2-agonists to children with acute asthma.

Pulmonary Epithelial Permeability in Hyaline-Membrane Disease

Abstract Neonatal hyaline-membrane disease is complicated by pulmonary edema, yet left atrial pressures are normal. Alveolar-capillary-membrane permeability may therefore be increased. To assess pulmonary epithelial permeability, we measured the pulmonary clearance and half-life of aerosolized 99mTc-diethylenetriamine pentacetate (99mTc-DTPA) on 31 occasions in 15 intubated premature infants with hyaline-membrane disease. Three infants with respiratory failure due to other diseases were studied on four occasions. All studies of infants with hyaline-membrane disease that were performed in the first 72 hours of life demonstrated a biphasic clearance curve with a rapid-phase half-life of 1.6±0.6 minutes (mean ±S.D.). As these infants recovered, the curve became monophasic with a half-life of 56.0±32.1 minutes. Two infants remained dependent on oxygen and ventilator support and had persistent biphasic curves with a rapid-phase half-life of 1.5±0.7 minutes. All infants without hyaline-membrane disease had mono...

Lung disease in mice with cystic fibrosis.

The leading cause of mortality and morbidity in humans with cystic fibrosis is lung disease. Advances in our understanding of the pathogenesis of the lung disease of cystic fibrosis, as well as development of innovative therapeutic interventions, have been compromised by the lack of a natural animal model. The utility of the CFTR-knockout mouse in studying the pathogenesis of cystic fibrosis has been limited because of their failure, despite the presence of severe intestinal disease, to develop lung disease. Herein, we describe the phenotype of an inbred congenic strain of CFTR-knockout mouse that develops spontaneous and progressive lung disease of early onset. The major features of the lung disease include failure of effective mucociliary transport, postbronchiolar over inflation of alveoli and parenchymal interstitial thickening, with evidence of fibrosis and inflammatory cell recruitment. We speculate that the basis for development of lung disease in the congenic CFTR-knockout mice is their observed lack of a non-CFTR chloride channel normally found in CFTR-knockout mice of mixed genetic background.

Regulation of an amiloride‐sensitive Na+‐permeable channel by a β2‐adrenergic agonist, cytosolic Ca2+ and Cl− in fetal rat alveolar epithelium

1 In cell‐attached patches formed on the apical membrane of fetal alveolar epithelium, terbutaline (a specific β2‐adrenergic agonist) increased the open probability (Po) of an amiloride‐sensitive Na+‐permeable non‐selective cation (NSC) channel (control, 0.03 ± 0.04; terbutaline, 0.62 ± 0.18; n= 8, P < 0.00001) by increasing the mean open time 100‐fold without any significant change in the mean closed time and without any change in the single channel conductance (control, 27.8 ± 2.3 pS; terbutaline, 28.2 ± 2.1 pS; n= 8). 2 The Po of the unstimulated channel increased when the apical membrane was depolarized due to a decrease in the closing rate and an increase in the opening rate, while the Po of the terbutaline‐stimulated channel did not depend on the membrane potential. 3 Increased cytosolic [Ca2+] also increased the Po of the channel in a manner consistent with one Ca2+‐binding site on the cytosolic surface of the channel. Terbutaline increased the sensitivity of the channel to cytosolic Ca2+ by shifting the concentration of cytosolic Ca2+ ([Ca2+]c) required for half‐maximal activation to a lower [Ca2+]c value, leading to an increase in Po. 4 An increase in the cytosolic Cl− concentration ([Cl−]c) decreased the Po of the channel consistent with two Cl−‐binding sites by increasing the closing rate without any significant change in the opening rate. Terbutaline increased Po by reducing the effect of cytosolic Cl− to promote channel closing. 5 Taken together, these observations indicate that terbutaline activates a Ca2+‐activated, Cl−‐inhibitable, amiloride‐sensitive, Na+‐permeable NSC channel in fetal rat alveolar epithelium in two ways: first, through an increase in Ca2+ sensitivity, and second, through a reduction in the effect of cytosolic Cl− to promote channel closing.

Cation selective channel in fetal alveolar type II epithelium.

A cation selective channel was identified in the apical membrane of fetal rat (Wistar) alveolar type II epithelium using the patch clamp technique. The single channel conductance was 23 +/- 1.2 pS (n = 16) with symmetrical NaCl (140 mM) solution in the bath and pipette. The channel was highly permeable to Na+ and K+ (PNa/PK = 0.9) but essentially impermeant to chloride and gluconate. Membrane potential did not influence open state probability when measured in a high Ca2+ (1.5 mM) bath. The channel reversibly inactivated when the bath was exchanged with a Ca(2+)-free (less than 10(-9) M) solution. The Na+ channel blocker amiloride (10(-6) M) applied to the extracellular side of the membrane reduced P(open) relative to control patches; P(control) = 0.57 +/- 0.11 (n = 5), P(amiloride) = 0.09 +/- 0.07 (n = 4, p less than 0.01), however, amiloride did not significantly influence channel conductance (g); g(control) 19 +/- 0.9 pS (n = 5), 18 +/- 3.0 pS (n = 4). More than one current level was observed in 42% (16/38) of active patches; multiple current levels (ranging from 2 to 6) were of equal amplitude suggesting the presence of multiple channels or subconductance states. Channel activity was also evident in cell attached patches. Since monolayers of these cells absorb Na+ via an amiloride sensitive transport mechanism we speculate that this amiloride sensitive cation selective channel is a potential apical pathway for electrogenic Na+ transport in the alveolar region of the lung.

Covalent Antithrombin-Heparin Complexes with High Anticoagulant Activity INTRAVENOUS, SUBCUTANEOUS, AND INTRATRACHEAL ADMINISTRATION

Although heparin has been used clinically for prophylaxis and treatment of thrombosis, it has suffered from problems such as short duration within compartments in vivo that require long term anticoagulation. A covalent antithrombin-heparin complex has been produced with high anticoagulant activity and a long half-life relative to heparin. The product had high anti-factor Xa and antithrombin activities compared with noncovalent mixtures of antithrombin and heparin (861 and 753 units/mg versus 209 and 198 units/mg, respectively). Reaction with thrombin was rapid with bimolecular and second order rate constants of 1.3 × 109 m −1 s−1 and 3.1 × 109 m −1s−1, respectively. The intravenous half-life of the complex in rabbits was 2.6 h as compared with 0.32 h for similar loads of heparin. Subcutaneous injection of antithrombin-heparin resulted in plasma levels (peaking at 24–30 h) that were still detectable 96 h post-injection. Given the increased lifetime in these vascular and intravascular spaces, use of the covalent complex in the lung was investigated. Activity of antithrombin-heparin instilled into rabbit lungs remained for 48 h with no detection of any complex systemically. Thus, this highly active agent has features required for pulmonary sequestration as a possible treatment for thrombotic diseases such as respiratory distress syndrome.

Effects of exercise on lung lymph flow in sheep and goats during normoxia and hypoxia.

Vigorous exercise causes a marked increase in cardiac output with only a minimal increase in measureable pulmonary vascular pressures. These changes in pulmonary hemodynamics should affect lung water and solute movement. On nine occasions, we measured the effect of normoxic exercise on lung lymph flow in four sheep and two goats with chronic lymph fistulas (wt = 15-25 kg). In addition, lymph flow was also measured on five occasions in sheep during exercise at reduced barometric pressures (430 and 380 mmHg). During normobaria, the animals ran at 3-5 km/h with 0-10% elevation of the treadmill for 15 to 85 min. Exercise on average caused a 100% increase in cardiac output, a 140% increase in lung lymph flow, and a slight but significant reduction in lymph to plasma concentration ratio (l/p) for total protein and albumin (mol wt = 70,000). There was a significant linear correlation between lymph flow and cardiac output (r = 0.87, P less than 0.01). There was no change in l/p for IgG (mol wt = 150,000) or IgM (mol wt = 900,000) and no significant change in mean pulmonary arterial (Ppa) or mean left atrial (Pla) pressures. Transition from normobaria to hypobaria caused an increase in Ppa but no change in Pla, cardiac output, or lymph flow. Exercise during hypobaria caused increases in lymph flow that were qualitatively similar to changes observed during normobaric exercise: there was a 60% increase in cardiac output, a 90% increase in lymph flow, and an 11% reduction in l/p for total protein. There was no change in l/p for albumin, IgG, or IgM, and no further change in Ppa. The increased lymph flow during normoxic and hypobaric exercise is best explained by an increase in pulmonary vascular surface area for fluid and protein exchange. Our results suggest that the normal ovine lung has the potential to nearly triple the amount of perfused microvascular surface area. This speculation is relevant to the interpretation of lymph flow data from other experiments.

Inhibition of amiloride-sensitive sodium-channel activity in distal lung epithelial cells by nitric oxide

Distal lung epithelial cells (DLECs) play an active role in fluid clearance from the alveolus by virtue of their ability to actively transport Na+ from the alveolus to the interstitial space. The present study evaluated the ability of activated macrophages to modulate the bioelectric properties of DLECs. Low numbers of lipopolysaccharide (LPS)-treated macrophages were able to significantly reduce amiloride-sensitive short-circuit current ( I sc) without affecting total I sc or monolayer resistance. This was associated with a rise in the flufenamic acid-sensitive component of the I sc. The effect was reversed by the addition of N-monomethyl-l-arginine to the medium, implying a role for nitric oxide. We hypothesized that macrophages exerted their effect by expressing inducible nitric oxide synthase (iNOS) in DLECs. The products of LPS-treated macrophages increased the levels of iNOS protein and mRNA transcripts in DLECs as well as causing a rise in iNOS activity. Immunofluorescence microscopy of LPS-stimulated macrophage-DLEC cocultures with anti-nitrotyrosine antibodies provided evidence for the generation of peroxynitrite in macrophages but not in DLECs. These data indicate that activated macrophages in the lung may contribute to impaired resolution of acute respiratory distress syndrome and suggest a novel mechanism whereby nitric oxide might alter cell function by altering its ion-transporting phenotype.Distal lung epithelial cells (DLECs) play an active role in fluid clearance from the alveolus by virtue of their ability to actively transport Na+ from the alveolus to the interstitial space. The present study evaluated the ability of activated macrophages to modulate the bioelectric properties of DLECs. Low numbers of lipopolysaccharide (LPS)-treated macrophages were able to significantly reduce amiloride-sensitive short-circuit current (Isc) without affecting total Isc or monolayer resistance. This was associated with a rise in the flufenamic acid-sensitive component of the Isc. The effect was reversed by the addition of N-monomethyl-L-arginine to the medium, implying a role for nitric oxide. We hypothesized that macrophages exerted their effect by expressing inducible nitric oxide synthase (iNOS) in DLECs. The products of LPS-treated macrophages increased the levels of iNOS protein and mRNA transcripts in DLECs as well as causing a rise in iNOS activity. Immunofluorescence microscopy of LPS-stimulated macrophage-DLEC cocultures with anti-nitrotyrosine antibodies provided evidence for the generation of peroxynitrite in macrophages but not in DLECs. These data indicate that activated macrophages in the lung may contribute to impaired resolution of acute respiratory distress syndrome and suggest a novel mechanism whereby nitric oxide might alter cell function by altering its ion-transporting phenotype.