Career development and compensation: strategies for physicians in academic health science centers. A perspective from a Canadian academic health science center.
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Academic Medical Centers↗
Biomedical subjects
H O'Brodovich
Publications and source records attributed to H O'Brodovich.
Translational activation and repression by distinct elements within the 5'-UTR of ENaC alpha-subunit mRNA.
The rat amiloride-sensitive epithelial Na(+) channel (rENaC), the rate-limiting step in epithelial Na(+) transport, consists of three subunits, alpha, beta, and gamma. We hypothesized that alpha-rENaC translation is regulated via its 5'-untranslated region (UTR). Transient transfections of alpha-rENaC promoter-reporter constructs in representative epithelial cell lines demonstrated up to fivefold differences in activity among constructs containing different amounts of the alpha-rENaC 5'-UTR sequence. Differences in reporter protein activity did not parallel differences in reporter mRNA, demonstrating that 5'-UTR regulation must be at the level of translation. Specifically, translation was enhanced by a region extending from +53 to +211 bp downstream from the transcription start site and repressed by the region between +367 and +499 bp. Examination of the 5'-UTR sequence revealed an out-of-frame initiation codon within the repressive region, 43 bp upstream from the start of the alpha-rENaC open reading frame. Mutational analysis of this upstream start codon indicated that it plays, at most, a minor role in impeding translation both in vitro and in vivo, suggesting that additional mechanisms of translational regulation are operative.
5' Untranslated Regions↗
Pulmonary edema fluid movement within the lung.
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Animals↗
Expression of alpha-, beta-, and gamma-hENaC mRNA in the human nasal, bronchial, and distal lung epithelium.
The amount of fluid covering the epithelium of the airways and alveolar space is modulated by active transport of Na+ from the lumen through the apical membrane Na+ permeant ion channels towards the interstitial space. We have measured the subunit expression of the amiloride-sensitive human Na+ channel (hENaC) by concomitant assessment of alpha-, beta-, and gamma-hENaC mRNA in the nasal, bronchial, and peripheral lung epithelia of adult patients undergoing lobectomy secondary to lung cancer. The study employed quantitative competitive reverse-transcriptase-polymerase chain reaction and qualitative in situ hybridization techniques. The hENaC mRNA content of each sample was normalized to the amount of epithelial cell-specific cytokeratin 18 (CK18) mRNA. Nasal epithelium contained significantly more (p < 0.05) alpha-hENaC mRNA (18 +/- 5 SD amol/fmol CK18), than bronchus (8 +/- 2 SD amol/fmol) and peripheral lung (9 +/- 2 SD amol/fmol). The ratio of gamma-hENaC/alpha-hENaC mRNA concentration was lowest in the nasal area, and it increased significantly towards the distal lung regions. The change in beta-hENaC mRNA was less profound. In situ hybridization studies of bronchial and peripheral lung sections selectively revealed expression of alpha-hENaC mRNA in superficial epithelium and submucosal glands of large airways, in bronchiolar epithelium, and in alveolar cells. We conclude that the relative expression of the hENaC subunit genes changes from the proximal to distal regions of the human respiratory tract.
Aged↗
Fetal lung liquid secretion: insights using the tools of inhibitors and genetic knock-out experiments.
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Animals↗
Transplant immunosuppression increases and prolongs transgene expression following adenoviral-mediated transfection of rat lungs.
BACKGROUND: Gene therapy provides the potential to modify donor organs to better withstand transplantation, but this has yet to be realized. In vivo gene transfer using adenoviral vectors has had limited success because of host immune response that induces inflammation and limits the amount and duration of transgene expression. We hypothesize that transplantation immunosuppression can attenuate the post-transfection host-immune response to allow for improved gene transfer following adenoviral-mediated transfection. METHODS: We intratracheally transfected with adenovirus containing the beta-galactosidase gene and randomized the rats to either the immunosuppression group, receiving daily cyclosporine, azathioprine, and methylprednisolone, or the control group, receiving no immunosuppression. We evaluated transgene expression and post-transfection inflammation at time points ranging from 1 day to 5 weeks. RESULTS: Following transfection, control rats showed relatively low levels of transgene expression, which rapidly decreased to non-detectable levels. In contrast, immunosuppressed rats demonstrated significantly higher levels of transgene expression overall (p < 0.00005), peaking at almost 3 times that of the control group (p < 0.02), and showing prolonged and elevated transgene expression at 5 weeks (p < 0.02). On histologic sections of the lungs, immunosuppressed rats exhibited overall lesser grades of post-transfection inflammation. CONCLUSIONS: Transplant immunosuppression provides the means to attenuate the severe immune response to adenoviral-mediated gene transfection and thereby increase and prolong transgene expression.
Adenoviridae↗
Oxygen induction of epithelial Na(+) transport requires heme proteins.
Fetal distal lung epithelial (FDLE) cells exposed to a postnatal O(2) concentration of 21% have higher epithelial Na(+) channel (ENaC) mRNA levels and Na(+) transport relative to FDLE cells grown in a fetal O(2) concentration of 3%. To investigate the mechanism of this process, FDLE monolayers were initially cultured in 3% O(2), and then some were switched to a 21% O(2) environment. Incubation of FDLE cells with the iron chelator deferoxamine, CoCl(2), NiCl(2), or an inhibitor of heme synthesis prevented or diminished the O(2) induction of amiloride-sensitive short-circuit current in FDLE cells. Similarly, defer- oxamine and cobalt prevented O(2)-induced ENaC mRNA expression. Exposure of FDLE cells grown under hypoxic conditions to carbon monoxide increased both ENaC mRNA expression and amiloride-sensitive short-circuit current. We therefore concluded that induction of ENaC mRNA expression and amiloride-sensitive Na(+) transport in FDLE cells by a physiological increase in O(2) concentration seen at birth requires iron and heme proteins.
Amiloride↗
Preventing endotoxin-stimulated alveolar macrophages from decreasing epithelium Na+ channel (ENaC) mRNA levels and activity.
The acute respiratory distress syndrome is characterized by impairment of the alveolar-capillary barrier. Our laboratory has shown that distal lung epithelial cell (DLEC) amiloride-sensitive Na+ transport is impaired by in vitro coculture with endotoxin (lipopolysaccharide)-stimulated alveolar macrophages (AM) through an L-arginine-dependent mechanism. To investigate the effect of this model on mRNA levels of the rat epithelial Na+ channel, mature fetal rat DLEC monolayers were incubated for 16 h with rat AM (1 x 10(7)) and lipopolysaccharide (10 microg/mL), or the cell-free supernatant of lipopolysaccharide-stimulated rat AM. Such exposure resulted in a profound decrease in mRNA expression for all subunits (alpha, beta, and gamma) of the rat epithelial Na+ channel, without affecting 18S RNA levels. This effect was prevented by the antioxidant N-acetylcysteine. In separate experiments, confluent DLEC monolayers were exposed to lipopolysaccharide-stimulated AM supernatant for 16 h with or without N-acetylcysteine and DTT and studied in Ussing chambers. As previously demonstrated in our laboratory, AM supernatant resulted in a significant (p < 0.05) impairment of DLEC Na+ transport, as reflected by a decrease in the amiloride-sensitive component of short-circuit current (control, 3.96 +/- 0.18 microA/cm2 versus supernatant, 2.34 +/- 0.56 microA/cm2; p < 0.05). This effect was significantly reversed by N-acetylcysteine (3.55 +/- 0.48 microA/cm2), but not by DTT (1.87 +/- 0.21 microA/cm2). N-acetylcysteine, but not DTT, increased DLEC thiol levels. These studies elucidate mechanisms by which activated AM impair alveolar epithelial barrier function in an in vitro model of acute lung injury.
Animals↗
Regulation of an amiloride-sensitive Na+-permeable channel by a beta2-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 beta2-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.
Adrenergic beta-Agonists↗
Roles of Ca2+ and protein tyrosine kinase in insulin action on cell volume via Na+ and K+ channels and Na+/K+/2Cl- cotransporter in fetal rat alveolar type II pneumocyte.
The aim of the present study was to investigate the roles of Ca2+ and protein tyrosine kinase (PTK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocytes. Insulin (100 nm) increased cell volume in the presence of extracellular Ca2+ (1 mm), while cell shrinkage was induced by insulin in the absence of extracellular Ca2+ (<1 nm). This insulin action in a Ca2+-containing solution was completely blocked by co-application of bumetanide (50 microm, an inhibitor of Na+/K+/2Cl- cotransporter) and amiloride (10 microm, an inhibitor of epithelial Na+ channel), but not by the individual application of either bumetanide or amiloride. On the other hand, the insulin action on cell volume in a Ca2+-free solution was completely blocked by quinine (1 mm, a blocker of Ca2+-activated K+ channel), but not by bumetanide and/or amiloride. These observations suggest that insulin activates an amiloride-sensitive Na+ channel and a bumetanide-sensitive Na+/K+/2Cl- cotransporter in the presence of 1 mm extracellular Ca2+, that the stimulatory action of insulin on an amiloride-sensitive Na+ channel and a bumetanide-sensitive Na+/K+/2Cl- cotransporter requires Ca2+, and that in a Ca2+-free solution insulin activates a quinine-sensitive K+ channel but not in the presence of 1 mm Ca2+. The insulin action on cell volume in a Ca2+-free solution was almost completely blocked by treatment with BAPTA (10 microm) or thapsigargin (1 microM, an inhibitor of Ca2+-ATPase which depletes the intracellular Ca2+ pool). Further, lavendustin A (10 microm, an inhibitor of receptor type PTK) blocked the insulin action in a Ca2+-free solution. These observations suggest that the stimulatory action of insulin on a quinine-sensitive K+ channel is mediated through PTK activity in a cytosolic Ca2+-dependent manner. Lavendustin A, further, completely blocked the activity of the Na+/K+/2Cl- cotransporter in a Ca2+-free solution, but only partially blocked the activity of the Na+/K+/2Cl- cotransporter in the presence of 1 mm Ca2+. This observation suggests that the activity of the Na+/K+/2Cl- cotransporter is maintained through two different pathways; one is a PTK-dependent, Ca2+-independent pathway and the other is a PTK-independent, Ca2+-dependent pathway. Further, we observed that removal of extracellular Ca2+ caused cell shrinkage by diminishing the activity of the amiloride-sensitive Na+ channel and the bumetanide-sensitive Na+/K+/2Cl- cotransporter, and that removal of extracellular Ca2+ abolished the activity of the quinine-sensitive K+ channel. We conclude that the cell shrinkage induced by removal of extracellular Ca2+ results from diverse effects on the cotransporter and Na+ and K+ channels.
Amiloride↗
HSC clinical trials controversy continues. Hospital for Sick Children.
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Biomedical Research↗
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 70,000) 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.
Blotting, Southern↗
Alternate funding arrangements to compensate physicians at academic health science centers.
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Academic Medical Centers↗
Sodium channels in alveolar epithelial cells: molecular characterization, biophysical properties, and physiological significance.
At birth, fetal distal lung epithelial (FDLE) cells switch from active chloride secretion to active sodium (Na+) reabsorption. Sodium ions enter the FDLE and alveolar type II (ATII) cells mainly through apical nonselective cation and Na(+)-selective channels, with conductances of 4-26 pS (picoSiemens) in FDLE and 20-25 pS in ATII cells. All these channels are inhibited by amiloride with a 50% inhibitory concentration of < 1 microM, and some are also inhibited by [N-ethyl-N-isopropyl]-2'-4'-amiloride (50% inhibitory concentration of < 1 microM). Both FDLE and ATII cells contain the alpha-, beta-, and gamma-rENaC (rat epithelial Na+ channels) mRNAs; reconstitution of an ATII cell Na(+)-channel protein into lipid bilayers revealed the presence of 25-pS Na+ single channels, inhibited by amiloride and [N-ethyl-N-isopropyl]-2'-4'-amiloride. A variety of agents, including cAMP, oxygen, glucocorticoids, and in some cases Ca2+, increased the activity and/or rENaC mRNA levels. The phenotypic properties of these channels differ from those observed in other Na(+)-absorbing epithelia. Pharmacological blockade of alveolar Na+ transport in vivo, as well as experiments with newborn alpha-rENaC knock-out mice, demonstrate the importance of active Na+ transport in the reabsorption of fluid from the fetal lung and in reabsorbing alveolar fluid in the injured adult lung. Indeed, in a number of inflammatory diseases, increased production of reactive oxygen-nitrogen intermediates, such as peroxynitrite (ONOO-), may damage ATII and FDLE Na+ channels, decrease Na+ reabsorption in vivo, and thus contribute to the formation of alveolar edema.
Animals↗
Structure and hormone responsiveness of the gene encoding the alpha-subunit of the rat amiloride-sensitive epithelial sodium channel.
The rat amiloride-sensitive epithelial sodium channel (rENaC) is the rate-limiting step for vectorial transport of Na+ across tight epithelia. The complex is composed of three subunits, alpha, beta, and gamma. Expression of the subunits has been shown to be tissue-specific and developmentally and hormonally regulated. To study mechanisms involved in transcriptional regulation of alpharENaC, we determined the genomic organization of the alpharENaC gene. By 5' rapid amplification of cDNA ends and primer extension, two transcriptional start sites were detected 453 base pairs (bp) apart, resulting in alternative 5' untranslated region (UTR) lengths of 515 or 62 bp. The longer 5' UTR is more prevalent in fetal lung than in adult lung or kidney. The 5' untranslated and coding regions are contained within 12 exons, with the translation start site located within the first exon. Sequence analysis of approximately 1,500 bp of 5' flanking DNA identified putative binding sites for transcription factors PEA3, SP1, AP-1, nuclear factor-kappaB, and thyroid and glucocorticoid receptors. alpharENaC promoter-reporter gene constructs produced low levels of reporter gene activity in transiently transfected cells, which could be increased by dexamethasone (DEX) treatment. Tri-iodothyronine treatment alone had no effect but potentiated stimulation by DEX.
Amino Acid Sequence↗
Hormonal regulation and genomic organization of the human amiloride-sensitive epithelial sodium channel alpha subunit gene.
To investigate the regulation of the amiloride-sensitive epithelial sodium channel (ENaC) expression, we have characterized the genomic structure and performed promoter analyses of the alpha subunit of the human (h) ENaC gene. Genomic clones containing the alphahENaC gene were isolated and subjected to restriction-mapping analysis. The alphahENaC gene was shown to be composed of 13 exons and 12 introns. Primer extension analysis confirmed that transcription initiation occurred at the beginning of the reported alphahENaC cDNA, but also indicated potential heterogenous initiation sites. Examination of a 3.1 kb 5' flanking sequence revealed a notable absence of CCAAT or TATA-like elements but suggested three GC boxes and several putative transcription factor binding sites, including a glucocorticoid response element (GRE) consensus. A 250 bp minimal promoter was capable of directing expression of a secreted alkaline phosphatase reporter. This promoter activity was enhanced 2.5- and 4-fold by upstream flanking sequences. Dexamethasone treatment induced levels of expression from the longer, GRE-containing promoter fragments from 8- to 20-fold, but not from the minimal promoter. Precise deletion of the 15-bp, dyad GRE sequence completely abolishes the response of reporter expression to dexamethasone induction. These experiments indicate that glucocorticoid augmentation of lung epithelial Na+ transport occurs, at least in part, by direct stimulation of transcription of the ENaC genes.
Base Sequence↗
Investigation of the anticoagulant mechanisms of a covalent antithrombin-heparin complex.
Recently, we developed a covalent antithrombin-heparin complex (ATH) as a possible treatment for respiratory distress syndrome. ATH reacted rapidly with thrombin and efficiently catalyzed the inhibition of either thrombin or factor Xa by exogenous antithrombin. In order to investigate mechanisms for the conjugate's unusual anticoagulant properties, changes in fluorescence due to covalent linkage or addition of exogenous antithrombin were studied in relation to reaction with thrombin derivatives or factor Xa. The emission spectrum of ATH was similar to that of antithrombin plus heparin mixtures. ATH quickly inhibited thrombin or factor Xa activities, as measured by a fluorogenic substrate. Fluorescein-labeled heparin was displaced from either thrombin or active site blocked thrombin by ATH, indicating that thrombin must bind to the conjugate's heparin moiety. Interaction of thrombin with ATH's heparin component was confirmed by a slow reaction rate of conjugate with a thrombin mutant that has weak heparin binding. Total intrinsic fluorescence increased when exogenous antithrombin was added to ATH, indicating that the catalytic mechanism may occur through a second inhibitor binding site. Thus, ATH reacts directly with thrombin through a bridge mechanism and probably catalyzes the reaction of thrombin with antithrombin by a second binding sequence on its heparin chain.
Anticoagulants↗
Covalent heparin cofactor II-heparin and heparin cofactor II-dermatan sulfate complexes. Characterization of novel anticoagulants.
Heparin cofactor II is a naturally occurring anticoagulant that acts by specifically inhibiting thrombin and is facilitated by the binding of glycosaminoglycans such as heparin and dermatan sulfate. In vivo, heparin cofactor II-glycosaminoglycan complexes dissociate, leaving the inhibitor less active in its ability to function as a component of the anticoagulation pathway. We have produced permanently activated heparin cofactor II molecules by covalent linkage to either heparin or dermatan sulfate. Covalent heparin cofactor II-heparin and heparin cofactor II-dermatan sulfate complexes had catalytic antithrombin activities similar to those of the corresponding starting heparin and dermatan sulfate (86% and 110% of standard heparin and dermatan sulfate activity, respectively). Both heparin cofactor II-heparin and heparin cofactor II-dermatan sulfate had fast bimolecular rate constants of 1.4 x 10(7) M-1 s-1 and 1.3 x 10(7) M-1 s-1, respectively, for reaction with thrombin. The intravenous half-life of the covalent complexes in rabbits was significantly longer than that of free heparin or dermatan sulfate (4.4, 3.4, 0.33, and 0.50 h for heparin cofactor II-heparin, heparin cofactor II-dermatan sulfate, heparin, and dermatan sulfate, respectively). Given their unique properties, these conjugates may have a clinical application for long term, selective inhibition of thrombin.
Animals↗