PubMed Health⌕ Search

Biomedical subjects

L Tung

Publications and source records attributed to L Tung.

At least 19 recordsLinked to original sources
PubMed2000

Estrogen receptors alpha and beta: prevalence of estrogen receptor beta mRNA in human vascular smooth muscle and transcriptional effects.

BACKGROUND: Estrogens have vascular effects through the activation of estrogen receptors (ERs). In addition to ERalpha, the first ER to be cloned, a second subtype called ERbeta has recently been discovered. METHODS AND RESULTS: Using a reverse-transcriptase polymerase chain reaction assay that employs the same primer pair to simultaneously amplify ERalpha and ERbeta transcripts, we found that ERbeta is the ER form that is predominantly expressed in human vascular smooth muscle, particularly in women. The transcriptional effects of the 2 ERs in transfected HeLa cells differed. In response to 17beta-estradiol, ERalpha is a stronger transactivator than ERbeta at low receptor concentrations. However, at higher receptor concentrations, ERalpha activity self-squelches, and ERbeta is a stronger transactivator. Tamoxifen has partial agonist effects with ERalpha but not with ERbeta. CONCLUSIONS: The protective effects of estrogens in the cardiovascular system of women may be due to the genomic effects of ERbeta in vascular tissue.

Estradiol↗
PubMed2000

Contact fluorescence imaging of reentry in monolayers of cultured neonatal rat ventricular myocytes.

INTRODUCTION: We present a novel contact fluorescence imaging (CFI) approach to monitor transmembrane potentials in monolayers of cultured neonatal rat ventricular cells. We apply CFI to demonstrate, for the first time, long-term recordings as well as electrical induction and termination of reentrant activity in this in vitro model. METHODS AND RESULTS: CFI was performed in confluent cell monolayers stained with di-8-ANEPPS. An anatomic obstacle (6 x 0.5 mm) was created in the center of the monolayers. Reentry was induced with a premature stimulus after pacing at 2 Hz (both via field stimulation). Seven sustained (>3 min) reentrant episodes, anchored to the anatomic obstacle, were observed in three monolayers. Field stimulation (30 V/cm) was applied to successfully terminate 6 of the 7 reentries. Analysis of reentrant activity showed similarities with anatomic reentry in tissue preparations, such as reduced conduction velocity around the core, variable conduction velocity along the reentrant pathway due to wavefront curvature effects, and field-induced activation at the obstacle borders leading to reentry termination (cardioversion). CONCLUSION: This study demonstrates the feasibility of CFI for macroscopic optical mapping of transmembrane potentials in a single layer of cultured cells. Our results suggest that the monolayer cell culture model is an attractive complement to tissue models of reentry and cardioversion.

Animals↗
PubMed2000

Focal extracellular potential: a means to monitor electrical activity in single cardiac myocytes.

The focal extracellular potential (FEP) described in this study is an electrophysiological signal related to the transmembrane potential (V(m)) of cardiac myocytes that avoids the mechanical fragility, interference with contraction, and intracellular contact associated with conventional whole cell recording. One end of a frog ventricular myocyte was secured into a glass holding pipette. The FEP was measured differentially between this pipette and a bath pipette while the cell was voltage- or current-clamped by a third whole cell pipette. The FEP appeared as an amplitude-truncated action potential, while FEP duration accurately reflected the action potential duration (APD) at 90% repolarization (APD(90)). FEP magnitude increased as the holding pipette K(+) concentration ([K(+)]) was increased. The FEP-voltage relation was quasi-linear at negative V(m) with a slope that increased with elevated holding pipette [K(+)]. Increasing the membrane conductance inside the holding pipette by adding amphotericin B or cromakalim linearized the FEP-voltage relation across all V(m). The FEP accurately reported electrical activation and APD(90) during changes of stimulation frequency and episodes of cellular stretch.

Action Potentials↗
PubMed2000

Association of the Ku autoantigen/DNA-dependent protein kinase holoenzyme and poly(ADP-ribose) polymerase with the DNA binding domain of progesterone receptors.

Ligand-activated progesterone receptors (PR) bind to DNA at specific progesterone response elements by means of a DNA binding domain (DBD(PR)) containing two highly conserved zinc fingers. DNA-bound PRs regulate transcription via interaction with other nuclear proteins and transcription factors. We have now identified four HeLa cell nuclear proteins that copurify with a glutathionine-S-transferase-human DBD(PR )fusion protein. Microsequence and immunoblot analyses identified one of these proteins as the 113 kDa poly(ADP-ribose) polymerase. The three other proteins were identified as subunits of the DNA-dependent protein kinase (DNA-PK) holoenzyme: its DNA binding regulatory heterodimers consisting of Ku70 and Ku86, and the 460 kDa catalytic subunit, DNA-PK(CS). DNA-PK that was 'pulled-down' by DBD(PR) on the affinity resin was able to (1) autophosphorylate Ku70, Ku86, and DNA-PK(CS), (2) transphosphorylate DBD(PR), and (3) phosphorylate a DNA-PK-specific p53 peptide substrate. DNA-PK was also able to associate with the DBD of the yeast activator GAL4. However, neither a PR DBD mutant lacking a structured first zinc finger (DBD(CYS)) nor the core DBD of the estrogen receptor (DBD(ER)) copurified DNA-PK, suggesting the interaction is not non-specific for DBDs. Lastly, we found that DNA-PK copurified with full-length human PR transiently expressed in HeLa cells, suggesting that the human PR/DNA-PK complex can assemble in vivo. These data show that DNA-PK and DBD(PR) interact, that DBD(PR) is a phosphorylation substrate of DNA-PK, and suggest a potential role for DNA-PK in PR-mediated transcription.

Amino Acid Sequence↗
PubMed1999

Changes in electroporation thresholds of lipid membranes by surfactants and peptides.

This article reviews recent work from our laboratory that explores how chemical additives may alter the threshold of electroporation of synthetic lipid bilayers. The addition of the nonionic block copolymeric surfactant, poloxamer 188 (P188), at a concentration of 1 mM increased the electroporation thresholds of planar lipid bilayer membranes made of azolectin. For a 10-microsecond rectangular pulse, P188-treated membranes were found to have a statistically higher threshold voltage, longer latency time to rupture, and lower postpulse conductance. Addition of the nonionic surfactant, octaethyleneglycol-mono-n-dodecyl-ether (C12E8), decreased the electroporation threshold of bilayer membranes made of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) by 10-40% for 10-microsecond- to 10-s-duration pulses, in a concentration-dependent manner over concentrations ranging from 0.1 to 10 mM. Postpulse membrane conductance also increased. The opposite effects of the two surfactants on electroporation thresholds may result from their very different structures, which would encourage different modes of surfactant-lipid interactions. To examine protein-lipid interactions and their effects on the electroporation threshold, the effects of a channel-forming polypeptide, gramicidin D (gD), was studied on membrane conductance and electroporation threshold. Electroporation thresholds for 15-ms pulses were unaffected by addition of gramicidin to POPC at a peptide:lipid concentration estimated to be 1:10,000, but increased significantly at ratios of 1:500 and 1:15, while membrane conductance increased monotonically with peptide concentration. A micropipette aspiration technique was applied to giant unilamellar POPC vesicles to measure changes in the membrane physical properties. When gD was added to give an estimated peptide:lipid ratio of 1:15, the membrane area expansivity modulus increased, indicating that the increase in electroporation threshold is correlated with a change in membrane stiffness. Thus, these findings demonstrate that surfactants or peptides can mediate the electroporation threshold of lipid bilayers.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1999

The effects of gramicidin on electroporation of lipid bilayers.

The effects of the channel-forming peptide gramicidin D (gD) on the conductance and electroporation thresholds of planar bilayer lipid membranes, made of the synthetic lipid 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC), was studied. High-amplitude ( approximately 200-900 mV) rectangular voltage pulses of 15 ms duration were used to perturb the bilayers and monitor the transmembrane conductance. Electroporation voltage thresholds were found, and conductance was recorded before and after electroporation. Gramicidin was added to the system in peptide/lipid ratios of 1:10, 000, 1:500, and 1:15. The addition of gD in a ratio of 1:10,000 had no effect on electroporation, but ratios of 1:500 and 1:15 significantly increased the thresholds by 16% (p < 0.0001) and 40% (p < 0.0001), respectively. Membrane conductance before electroporation was measurable only after the addition of gD and increased monotonically as the peptide/lipid ratio increased. The effect of gD on the membrane area expansivity modulus (K) was tested using giant unilamellar vesicles (GUVs). When gD was incorporated into the vesicles in a 1:15 ratio, K increased by 110%, consistent with the increase in thresholds predicted by an electromechanical model. These findings suggest that the presence of membrane proteins may affect the electroporation of lipid bilayers by changing their mechanical properties.

Biophysical Phenomena↗
PubMed1999

Separation between virtual sources modifies the response of cardiac tissue to field stimulation.

INTRODUCTION: While it is now understood that the tissue geometry and the electric field distribution are important in generating virtual electrodes, the effects of interaction between a collection of electrodes have not been examined. To develop a basis for understanding such interactions, we have studied a single pair of oppositely polarized virtual sources. Although such oppositely polarized pairs of virtual electrodes can be generated by a variety of field distributions and tissue geometries, we examine one simple system that incorporates the salient features of source interaction. METHODS AND RESULTS: Our model system is a homogeneous tissue strip stimulated by a uniform extracellular field. To clarify virtual source interaction, we show that field stimulated tissue can be equivalently polarized by a set of intracellular current sources with magnitude and distribution defined by the generalized activating function. In our model system, an intracellular current source is produced at one edge of the tissue and an intracellular current sink at the other. Therefore, the tissue length acts to modulate the overlap, or interaction, between the polarizations arising from each source. To quantify the effects of source interaction, the chronaxie and rheobase values of the strength-duration relation were determined for source separations varying between 1.0 cm and 100 microm (active membrane dynamics were modeled with the Luo-Rudy phase I formulation). At all separations >3.0 mm, the chronaxie was constant at 3.09 msec and the rheobase was 0.38 V/cm. Under 0.2 mm, the chronaxie decreased to 0.55 msec while the rheobase increased linearly with the inverse of source separation. The dependence of these parameters on separation primarily reflects passive electrotonic interactions between the two virtual electrodes. However, the exact values are strongly dependent upon active tissue properties-largely the inward rectifier potassium channel and activation of the sodium current. CONCLUSION: Tissue excitation in response to field stimulation is strongly modulated by the proximity of, and therefore the interaction between, oppositely polarized virtual electrode sources.

Computer Simulation↗
PubMed1999

Sequential change in action potential of rabbit epicardium during and following radiofrequency ablation.

INTRODUCTION: Although radiofrequency (RF) catheter ablation is used to treat certain cardiac arrhythmias, little is known regarding transient changes in cellular electrophysiology during and following RF delivery. Optical recordings of action potential (OAP) with voltage-sensitive dyes allow immunity from electrical noise during RF delivery. The purpose of this study was to clarify the possible synergistic effects of both the thermal and electrotonic components of RF ablation. METHODS AND RESULTS: In this study, OAPs were recorded on the epicardium of 16 isolated Langendorff-perfused rabbit hearts within or adjacent to lesions made by RF catheters. Hearts were perfused at room temperature with Tyrode's solution containing 2,3-butanedione monoxime and stained by the voltage-sensitive dye di-4-ANEPPS. OAPs were recorded before, during, and after RF pulses. Within the lesion, the action potential duration at 80% repolarization (APD80) of OAP decreased rapidly during the RF pulse, without recovery following the pulse. In the border zone surrounding the lesion, the RF energy resulted in a rapid decrease in APD80, which recovered promptly after the pulse (recovery time constant: 82 +/- 37 sec). APD80 was nonlinearly related to temperature during the RF ablation and responded faster to RF ablation than to purely thermal injury. CONCLUSION: The application of RF energy results in significant changes in myocardial cellular electrophysiologic properties. The RF energy has a combination of thermal and electrotonic effects on the myocardial tissue. The results of this in vitro study may illustrate the cellular basis for commonly observed phenomena in clinical practice.

Action Potentials↗
PubMed1999

Nonuniform responses of transmembrane potential during electric field stimulation of single cardiac cells.

The response of cellular transmembrane potentials (V(m)) to applied electric fields is a critical factor during electrical pacing, cardioversion, and defibrillation, yet the coupling relationship of the cellular response to field intensity and polarity is not well documented. Isolated guinea pig ventricular myocytes were stained with a voltage-sensitive fluorescent dye, di-8-ANEPPS (10 microM). A green helium-neon laser was used to excite the fluorescent dye with a 15-micrometers-diameter focused spot, and subcellular V(m) were recorded optically during field stimulation directed along the long axis of the cell. The membrane response was measured at the cell end with the use of a 30-ms S1-S2 coupling interval and a 10-ms S2 pulse with strength of up to approximately 500-mV half-cell length potential (field strength x one-half the cell length). The general trends show that 1) the response of V(m) at the cell end occurs in two stages, the first being very rapid (<1 ms) and the second much slower in time scale, 2) the rapid response consists of hyperpolarization when the cell end faces the anode and depolarization when the cell end faces the cathode, 3) the rapid response varies nonlinearly with field strengths and polarity, being relatively larger for the hyperpolarizing responses, and 4) the slower, time-dependent response has a time course that varies in slope with field strength. Furthermore, the linearity of the dye response was confirmed over a voltage range of -280 to +140 mV by simultaneous measurements of optically and electrically recorded V(m). These experimental findings could not be reproduced by the updated, Luo-Rudy dynamic model but could be explained with the addition of two currents that activate outside the physiological range of voltages: a hypothetical outward current that activates strongly at positive potentials and a second current that represents electroporation of the cell membrane.

Action Potentials↗
PubMed1999

Factors governing mechanical stimulation in frog hearts.

Because stretch-induced activation may be important in generating clinically relevant arrhythmias in the heart, we delineated the ability of different types of stretches to activate ventricular tissue. Geometrically simple sheets of frog (Rana catesbeiana) ventricular tissue were mounted to allow stretches to be applied perpendicular to one edge. Every heart could be activated by a stretch pulse (n = 25), and several parameters were varied to determine their effects on mechanical activation threshold. At shorter coupling intervals, a larger stretch was needed to excite the tissue, and activation-recovery intervals were shorter, similar to previously published electrically probed strength-interval and restitution relations. Additionally, the tissue became easier to activate as the speed of the stretch increased from 0.09 to 2.6% length/ms. The increment in stretch needed for activation decreased as the baseline stretch increased from 0 to 6% length. Thus we show that mechanical activation is similar to electrical activation and that increasing uniquely mechanical parameters such as the speed of the applied stretch or baseline level of stretch can decrease the mechanical activation threshold.

Animals↗
PubMed1998

Electroporation-mediated gene transfer in cardiac tissue.

Delivery of genes or macromolecules to cardiovascular tissues provides new therapeutic opportunities for the treatment of many acquired and inherited diseases. To investigate electroporation as a delivery method in cardiac tissue, embryonic chick hearts were studied for uptake of propidium iodide (PI) or DNA encoding either green fluorescent protein (GFP) or luciferase following electrical shock. PI uptake increased monotonically from 6% of heart tissue after 3 shocks to 77% with 12 shocks. GFP and luciferase expression varied in proportion to shock number, with detectable levels in all electrically treated hearts. Thus, electroporation promotes uptake of PI and DNA in cardiac tissue, suggesting further application of this method for therapeutic genes.

Animals↗
PubMed1998

Progesterone regulates transcription of the p21(WAF1) cyclin- dependent kinase inhibitor gene through Sp1 and CBP/p300.

Progesterone has biphasic effects on proliferation of breast cancer cells; it stimulates growth in the first cell cycle, then arrests cells at G1/S of the second cycle accompanied by up-regulation of the cyclin-dependent kinase inhibitor, p21. We now show that progesterone regulates transcription of the p21 promoter by an unusual mechanism. This promoter lacks a canonical progesterone response element. Instead, progesterone receptors (PRs) interact with the promoter through the transcription factor Sp1 at the third and fourth of six Sp1 binding sites located downstream of nucleotide 154. Mutation of Sp1 site 3 eliminates basal transcription, and mutation of sites 3 and 4 eliminates transcriptional up-regulation by progesterone. Progesterone-mediated transcription is further prevented by overexpression of E1A, suggesting that CBP/p300 is required. Indeed, in HeLa cells, Sp1 and CBP/p300 associate with stably integrated flag-tagged PRs in a multiprotein complex. Since many signals converge on p21, cross-talk between PRs and other factors co-localized on the p21 promoter, may explain how progesterone can be either proliferative or differentiative in different target cells.

Cyclin-Dependent Kinase Inhibitor p21↗
PubMed1998

An N-terminal inhibitory function, IF, suppresses transcription by the A-isoform but not the B-isoform of human progesterone receptors.

The B-isoform of human progesterone receptors (PR) contains three activation functions (AF3, AF1, and AF2), two of which (AF1 and AF2) are shared with the A-isoform. AF3 is in the B-upstream segment (BUS), the far N-terminal 164 amino acids of B-receptors; AF1 is in the 392-amino acid N-terminal region common to both receptors; and AF2 is in the C-terminal hormone binding domain. B-receptors are usually stronger transactivators than A-receptors due to transcriptional synergism between AF3 and one of the two downstream AFs. We now show that the N terminus of PR common to both isoforms contains an inhibitory function (IF) located in a 292-amino acid segment lying upstream of AF1. IF represses the activity of A-receptors but is not inhibitory in the context of B-receptors due to constraints imparted by BUS. As a result, IF inhibits AF1 or AF2 but not AF3, regardless of the position of IF relative to BUS. IF is functionally independent and strongly represses transcription when it is fused upstream of estrogen receptors. These data demonstrate the existence of a novel, transferable inhibitory function, mapping to the PR N terminus, which begins to assign specific roles to this large undefined region.

Gene Expression Regulation↗
PubMed1998

The reduction in electroporation voltages by the addition of a surfactant to planar lipid bilayers.

The effects of a nonionic surfactant, octaethyleneglycol mono n-dodecyl ether (C12E8), on the electroporation of planar bilayer lipid membranes made of the synthetic lipid 1-pamitoyl 2-oleoyl phosphatidylcholine (POPC), was studied. High-amplitude ( approximately 100-450 mV) rectangular voltage pulses were used to electroporate the bilayers, followed by a prolonged, low-amplitude ( approximately 65 mV) voltage clamp to monitor the ensuing changes in transmembrane conductance. The electroporation thresholds of the membranes were found for rectangular voltage pulses of given durations. The strength-duration relationship was determined over a range from 10 micros to 10 s. The addition of C12E8 at concentrations of 0.1, 1, and 10 microM to the bath surrounding the membranes decreased the electroporation threshold monotonically with concentration for all durations (p < 0.0001). The decrease from control values ranged from 10% to 40%, depending on surfactant concentration and pulse duration. For a 10-micros pulse, the transmembrane conductance 150 micros after electroporation (G150) increased monotonically with the surfactant concentration (p = 0.007 for 10 microM C12E8). These findings suggest that C12E8 incorporates into POPC bilayers, allowing electroporation at lower intensities and/or shorter durations, and demonstrate that surfactants can be used to manipulate the electroporation threshold of lipid bilayers.

Detergents↗
PubMed1998

Progesterone receptor variants found in breast cells repress transcription by wild-type receptors.

Progesterone, through its nuclear receptors (PR), regulates the development and growth of breast cancers. PR also serve as markers of hormone dependence and prognosis in patients with this disease, and functional PR are required to mediate the antiproliferative effects of progestin therapies. We find that normal and malignant breast cells and tissues can express anomalous forms of PR transcripts. We have isolated four variant PR mRNAs that contain precise deletions of exons encoding sections of the DNA- and hormone-binding domains. The transcripts lack exon 2 (PRdelta2), exon 4 (PRdelta4), exon 6 (PRdelta6), or exons 5 and 6 (PRdelta5,6). On immunoblots, PRdelta4, delta6. and delta5, 6 cloned into the background of the PR A-isoform comigrate with similar proteins present in breast tumor extracts; delta6 and delta5, 6 are dominant-negative transcriptional inhibitors of wild-type A- and B-receptors. We propose that expression of variant PR can compromise the accuracy of receptor measurements as markers of hormone-dependent cancers, and can modify the responses of tumors to progestin therapies.

Breast↗
PubMed1998

Postshock potential gradients and dispersion of repolarization in cells stimulated with monophasic and biphasic waveforms.

INTRODUCTION: Even though the clinical advantage of biphasic defibrillation waveforms is well documented, the mechanisms that underlie this greater efficacy remain incompletely understood. It is established, though, that the response of relatively refractory cells to the shock is important in determining defibrillation success or failure. We used two computer models of an isolated ventricular cell to test the hypothesis that biphasic stimuli cause a more uniform response than the equivalent monophasic shocks, decreasing the likelihood that fibrillation will be reinduced. METHODS AND RESULTS: Models of reciprocally polarized and uniformly polarized cells were used. Rapid pacing and elevated [K]o were simulated, and either 10-msec rectangular monophasic or 5-msec/5-msec symmetric biphasic stimuli were delivered in the relative refractory period. The effects of stimulus intensity and coupling interval on response duration and postshock transmembrane potential (Vm) were quantified for each waveform. With reciprocal polarization, biphasic stimuli caused a more uniform response than monophasic stimuli, resulting in fewer large gradients of Vm (only for shock strengths < or = 1.25x threshold vs < or = 2.125x threshold) and a smaller dispersion of repolarization (1611 msec2 vs 1835 msec2). The reverse was observed with uniform polarization: monophasic pulses caused a more uniform response than did biphasic stimuli. CONCLUSION: These results show that the response of relatively refractory cardiac cells to biphasic stimuli is less dependent on the coupling interval and stimulus strength than the response to monophasic stimuli under conditions of reciprocal polarization. Because this may lead to fewer and smaller spatial gradients in Vm, these data support the hypothesis that biphasic defibrillation waveforms will be less likely to reinduce fibrillation. Further, published experimental results correlate to a greater degree with conditions of reciprocal polarization than of uniform polarization, providing indirect evidence that interactions between depolarized and hyperpolarized regions play a role in determining the effects of defibrillation shocks on cardiac tissue.

Computer Simulation↗
PubMed1998

Stretch-induced changes in arrhythmogenesis and excitability in experimentally based heart cell models.

Mechanoelectric coupling in the heart is well documented and has been suggested as a cause of arrhythmia. One hypothesized mechanism for the stretch sensitivity of cardiac muscle is the presence of stretch-activated channels (SACs). This study uses modeling to explore the influence of SACs on cardiac resting potential, excitation threshold, and action potential in the context of arrhythmia. We added a putative SAC, modeled as a linear, time-independent conductance with reversal potential of -20 or -50 mV, to guinea pig and frog ventricular membrane models. Increased stretch conductance led to resting potential depolarization, a decreased excitation threshold, altered action potential duration, and, under certain conditions, early afterdepolarizations. We conclude that stretch increases cellular excitability, making the heart prone to ectopic activity. Regional effects of stretch on action potential duration can vary and are influenced by factors such as the SAC reversal potential, ionic conditions, and baseline currents, all of which may lead to an increased dispersion of refractoriness throughout the heart and therefore an increased risk of arrhythmia.

Action Potentials↗