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L Tung

Publications and source records attributed to L Tung.

At least 19 recordsLinked to original sources
PubMed2002

Cardiomyocyte cultures with controlled macroscopic anisotropy: a model for functional electrophysiological studies of cardiac muscle.

Structural and functional cardiac anisotropy varies with the development, location, and pathophysiology in the heart. The goal of this study was to design a cell culture model system in which the degree, change in fiber direction, and discontinuity of anisotropy can be controlled over centimeter-size length scales. Neonatal rat ventricular myocytes were cultured on fibronectin on 20-mm diameter circular cover slips. Structure-function relationships were assessed using immunostaining and optical mapping. Cell culture on microabraded cover slips yielded cell elongation and coalignment in the direction of abrasion, and uniform, macroscopically continuous, elliptical propagation with point stimulation. Coarser microabrasion (wider and deeper abrasion grooves) increased longitudinal (23.5 to 37.2 cm/s; r=0.66) and decreased transverse conduction velocity (18.1 to 9.2 cm/s; r=-0.84), which resulted in increased longitudinal-to-transverse velocity anisotropy ratios (1.3 to 3.7, n=61). A thin transition zone between adjacent uniformly anisotropic areas with 45 degrees or 90 degrees difference in fiber orientation acted as a secondary source during 2x threshold field stimulus. Cell culture on cover slips micropatterned with 12- or 25- micro m wide fibronectin lines and previously coated with decreasing concentrations of background fibronectin yielded transition from continuous to discontinuous anisotropic architecture with longitudinally oriented intercellular clefts, decreased transverse velocity (16.9 to 2.6 cm/s; r=-0.95), increased velocity anisotropy ratios (1.6 to 5.6, n=70), and decreased longitudinal velocity (36.4 to 14.6 cm/s; r=-0.85) for anisotropy ratios >3.5. Cultures of cardiac myocytes with controlled degree, uniformity and continuity of structural, and functional anisotropy may enable systematic 2-dimensional in vitro studies of macroscopic structure-related mechanisms of reentrant arrhythmias. The full text of this article is available at http://www.circresaha.org.

Action Potentials↗
PubMed2001

Mapping the unique activation function 3 in the progesterone B-receptor upstream segment. Two LXXLL motifs and a tryptophan residue are required for activity.

Progesterone receptors (PR) contain three activation functions (AFs) that together define the extent to which they regulate transcription. AF1 and AF2 are common to the two isoforms of PR, PR-A and PR-B, whereas AF3 lies within the N-terminal 164 amino acids unique to PR-B, termed the "B-upstream segment" (BUS). To define the BUS regions that contribute to AF3 function, we generated a series of deletion and amino acid substitution mutants and tested them in three backgrounds as follows: BUS alone fused to the PR DNA binding domain (BUS-DBD), the entire PR-B N terminus linked to its DBD (NT-B), and full-length PR-B. Analyses of these mutants identified two regions in BUS whose loss reduces AF3 activity by more than 90%. These are associated with amino acids 54-90 (R1) and 120-154 (R2). R1 contains a consensus (55)LXXLL(59) motif (L1) identical to ones found in nuclear receptor co-activators. R2 is adjacent to a second nuclear receptor box (L2) at (115)LXXLL(119) and contains a conserved tryptophan (Trp-140). Their mutation completely disrupts AF3 activity in a promoter and cell type-independent manner. Critical mutations elicited similar effects on all three B-receptor backgrounds. This underscores the probability that these mutations alter a process linking BUS structure to the function of full-length PR-B in a fundamental way.

Amino Acid Motifs↗
PubMed2001

Theoretical and experimental study of sawtooth effect in isolated cardiac cell-pairs.

INTRODUCTION: The question of how a defibrillation shock affects the myocardium far (> approximately 1 mm; the space constant of continuum tissue models) from the electrode is not fully understood. According to a long-standing, yet to be verified, hypothesis, the relatively high-resistance intercellular gap junctions may help in coupling the shock effect to the distant myocardium by redistributing the defibrillation current and creating a sawtooth pattern of polarization in which every cell undergoes hyperpolarization and depolarization. The goal of this study was to conduct an in-depth theoretical and experimental investigation of the sawtooth effect in the simplest coupled system, that of an isolated cell-pair. METHODS AND RESULTS: Theoretically, we present a relationship between sawtooth amplitude (STA) and junctional resistance (Rj), and show that, in a cell-pair with two cells of different lengths, the sawtooth effect may not necessarily appear as a reversal in polarization across the junction when Rj is below a critical value. Experimentally, we optically mapped transmembrane potential responses along the lengths of enzymatically isolated guinea pig cell-pairs at 10- or 17-microm resolution, and estimated STA as the magnitude of discontinuity in responses at the intercellular junction. From 14 cell-pairs, STA was estimated to be approximately 11 mV for a nominal 10 V/cm field. Based on our theoretical results, this value corresponds to an Rj of approximately 18 Mohms. CONCLUSION: The intercellular junction induces a measurable sawtooth effect in the simplest system of an isolated cell-pair. An accounting for the sawtooth effect might be essential for understanding field-tissue interaction far from the electrode and to accurately predict tissue response during field stimulation.

Animals↗
PubMed2000

Thoughts on tamoxifen resistant breast cancer. Are coregulators the answer or just a red herring?

The antiestrogen tamoxifen is an effective treatment for estrogen receptor positive breast cancers, slowing tumor growth and preventing disease recurrence, with relatively few side effects. However, many patients who initially respond to treatment, later become resistant to treatment. Tamoxifen has both agonist and antagonist activities, which are manifested in a tissue-specific pattern. Development of tamoxifen resistance can be characterized by an increase in the partial agonist properties of the antiestrogen in the breast, resulting in loss of growth inhibition and even inappropriate tumor stimulation. Nuclear receptor function is modulated by transcriptional coregulators, which either enhance or repress receptor activity. Using a mixed antagonist-biased two-hybrid screening strategy, we identified two such proteins: the human homolog of the nuclear receptor corepressor, N-CoR, and a novel coactivator, L7/SPA (Switch Protein for Antagonists). In transcriptional studies N-CoR suppressed the agonist properties of tamoxifen and RU486, while L7/SPA increased agonist effects. We speculated that the relative level of these coactivators and corepressors might determine the balance of agonist and antagonist properties of mixed antagonists such as tamoxifen. Using quantitative RT-PCR we therefore measured the levels of transcripts encoding these coregulators, as well as the corepressor SMRT, and the coactivator SRC-1, in a small cohort of tamoxifen resistant and sensitive breast tumors. The results suggest that tumor sensitivity to mixed antagonists may be governed by a complex set of transcription factors, which we are only now beginning to understand.

Animals↗
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

Virtual sources associated with linear and curved strands of cardiac cells.

Transmembrane potential (V(m)) responses in cardiac strands with different curvature were characterized during uniform electric-field stimulation with the use of modeling and experimental approaches. Linear and U-shaped strands (width 100-150 micrometer) were stained with voltage-sensitive dye. V(m) was measured by optical mapping across the width and at sites of beginning curvature. Field pulses were applied transverse to the strands during the action-potential plateau. For linear strands, V(m) contained 1) a rapid passive component (V(m)(ar)) nearly linear and symmetric across the width, 2) a slower hyperpolarizing component (V(m)(as)) greater and faster on the anodal side, and 3) at high field strengths a delayed depolarizing component (V(m)(ad)) greater on the anodal side. For U-shaped strands, V(m) at sites of beginning curvature also contained rapid and slow components (V(m)(br) and V(m)(bs), respectively) that included contributions from the linear strand response and from the fiber curvature. V(m)(ar), V(m)(br), and part of V(m)(bs) could be attributed to passive behavior that was modeled, and V(m)(as), V(m)(ad), and part of V(m)(bs) could be attributed to active membrane currents. Thus curved strands exhibit field responses separable into components with characteristic amplitude, spatial, and temporal signatures.

Animals↗
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↗