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Biomedical subjects

L Tung

Publications and source records attributed to L Tung.

At least 73 records · Page 4Linked to original sources
PubMed1991

Electroporation of cardiac cell membranes with monophasic or biphasic rectangular pulses.

During defibrillation, cardioversion, and electrocution trauma, heart cells are exposed to potential gradients that increase the transmembrane potential (Vm). At sufficiently high Vm, pathological increases in cell permeability can occur. With enzymatically isolated frog heart cells (n = 29) we investigated the voltage and time sufficient for electroporation or cardiac cell membranes with rectangular voltage pulses, particularly with 5-msec monophasic, and 5- or 10-msec biphasic pulses. The rectangular voltage pulse (monophasic 0.1-1.5 V, 0.1-100 msec or symmetric biphasic 0.1-1 V, 0.4-10 msec [total duration]) was applied to the cell membrane using the cell-attached patch clamp technique, and a low voltage pulse train was added so that membrane conductance could be monitored continuously. Step increases in membrane conductance (breakdown) were observed, indicative of electroporation, and occurred with different combinations of pulse amplitude and duration; for example, for monophasic square pulses: (1 V, 0.2 msec) or (0.5 V, 0.5 msec), and for biphasic pulses: (1 V, 0.4 msec total duration) or (0.5 V, 0.8 msec). Using 5- or 10-msec rectangular pulses, breakdown occurred at a voltage around 0.4 V independent of polarity or waveform. The recovery of the permeabilized cell membrane after the voltage pulse was highly variable, in some cases not recovering at all while in other cases recovering after a lapse of seconds to minutes. These results suggest that monophasic and biphasic pulses of approximately 1 V, 0.2-0.4 msec and approximately 0.4 V, 5 msec can permeabilize the heart cell membrane even for minutes, time enough to cause an alteration in the cellular ionic composition leading to depressed or unexcitable tissue, a precursor for cardiac arrhythmia.

Animals↗
PubMed1991

Cardiac mechanics at the cellular level.

The active mechanical properties of heart muscle are load, length, and time-dependent. The capability for investigating cardiac mechanisms at the cellular level may help to distinguish between those properties of the myocardium which arise from myocardial cells and those which arise from the tissue architecture and extracellular matrix of connective fibers. We present here, for the first time, a general approach for subjecting single heart cells to isometric, isotonic, afterloaded, or physiological loading sequences, while obtaining on-line measures of cell force and length. This approach has been implemented and tested on freshly dissociated, adult frog ventricular myocytes. Examples are presented for each of the four loading sequences.

Animals↗
PubMed1991

UHF-1, a factor required for maximal transcription of early and late sea urchin histone H4 genes: analysis of promoter-binding sites.

A protein, denoted UHF-1, was found to bind upstream of the transcriptional start site of both the early and late H4 (EH4 and LH4) histone genes of the sea urchin Strongylocentrotus purpuratus. A nuclear extract from hatching blastulae contained proteins that bind to EH4 and LH4 promoter fragments in a band shift assay and produced sharp DNase I footprints upstream of the EH4 gene (from -133 to -106) and the LH4 gene (from -94 to -66). DNase I footprinting performed in the presence of EH4 and LH4 promoter competitor DNAs indicated that UHF-1 binds more strongly to the EH4 site. A sequence match of 11 of 13 nucleotides was found within the two footprinted regions: [sequence: see text]. Methylation interference and footprinting experiments showed that UHF-1 bound to the two sites somewhat differently. DNA-protein UV cross-linking studies indicated that UHF-1 has an electrophoretic mobility on sodium dodecyl sulfate-acrylamide gels of approximately 85 kDa and suggested that additional proteins, specific to each promoter, bind to each site. In vitro and in vivo assays were used to demonstrate that the UHF-1-binding site is essential for maximal transcription of the H4 genes. Deletion of the EH4 footprinted region resulted in a 3-fold decrease in transcription in a nuclear extract and a 2.6-fold decrease in expression in morulae from templates that had been injected into eggs. In the latter case, deletion of the binding site did not grossly disrupt the temporal program of expression from the injected EH4 genes. LH4 templates containing a 10-bp deletion in the consensus region or base substitutions in the footprinted region were transcribed at 14 to 58% of the level of the wild-type LH4 template. UHF-1 is therefore essential for maximal expression of the early and late H4 genes.

Animals↗
PubMed1991

Influence of electrical axis of stimulation on excitation of cardiac muscle cells.

Orthogonal sequential shock can defibrillate the heart with greater efficacy compared with single shock defibrillation. In this study we tested the hypothesis that cardiac cells have a preferred orientation in their response to excitatory extracellular electric fields, so that orthogonal shocks may stimulate distinct populations of cells. A micropaddle electrode system was used to deliver rectangular pulses for extracellular field stimulation of individual heart cells. We found that single frog and guinea pig ventricular myocytes are excitable with rectangular pulse field stimulation over a wide range of pulse durations, ranging from 10 msec to as little as 20 microseconds. The excitation field strength varies inversely with pulse duration as described by the Weiss-Lapicque equation, although the frog myocytes show a significant "notch" at pulse durations of approximately 1-2 msec, and the guinea pig myocytes are more excitable than predicted for pulse durations of less than 0.2 msec. Every myocyte tested was more excitable when the long axis of the cell was oriented parallel to the stimulating field than when perpendicular to the field. For 2-msec pulses, the difference in field strength was a factor of 5.8 +/- 2.0 (n = 30) for frog and 2.6 +/- 0.5 (n = 23) for guinea pig myocytes. Complete excitation strength-duration curves were obtained in seven frog and 14 guinea pig cells for both parallel and perpendicular cell orientations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Positive and negative transcriptional regulatory elements in the early H4 histone gene of the sea urchin, Strongylocentrotus purpuratus.

The early H4 (EH4) histone gene of the sea urchin, Strongylocentrotus purpuratus, is shown to contain at least five positive-responding sequence elements and one negative-responding site which control the level of in vitro transcription in an embryonic nuclear extract. The positive acting elements are: 1) the UHF-1 region, located between -133 and -102 (the site of a strong footprint, due at least in part to the binding of an 85 kD protein factor termed UHF-1); 2) the H4 specific element (H4SE), situated between -62 and -39; 3) a sequence corresponding to a TATA box between -33 and -26 (TAACAATA); 4) the transcriptional initiation site; and 5) an internal sequence element found between +19 and +50. Deletion of, or base changes in, the UHF-1, H4SE, initiation, or internal sequence sites resulted in significant decreases in transcription. Base substitutions in the TATA-like sequence had much less effect, resulting in no more than a 2-fold decrease in transcription, and there was no evidence that alternative initiation sites are utilized in the mutant templates. The negative element (termed the UHF-3 site) is contained within a footprinted region between nucleotides -75 and -56. Base substitutions in this area result in templates that were transcribed at a level 1.2-2.0-fold higher than the wild-type gene. Transcription levels of double UHF-1/H4SE and UHF-1/INR mutants were those expected from additive effects of the individual mutations and there was no suggestion of synergism.

Animals↗
PubMed1990

Expression, purification and characterization of a 41 kDa insulin receptor tyrosine kinase domain.

An active 41 kDa cytoplasmic domain of the insulin receptor tyrosine kinase (CIRK-41) encompassing amino acid residues 946 and 1303 of the native protein with an additional three amino acids (HAI) at the N-terminus has been overexpressed using the baculovirus pAC 373 expression system. The recombinant protein termed CIRK-41 has been purified to homogeneity. CIRK-41 was capable of autophosphorylation and up to 1.9 moles of phosphate were incorporated per mole of enzyme when it was incubated in the presence of 10 mM manganese chloride and 0.5 mM ATP. Autophosphorylation resulted in stimulation of CIRK-41 activity towards its exogenous substrate, indicating that CIRK-41 may be used as a model molecule to study the role of phosphorylation and dephosphorylation in the control of the insulin receptor tyrosine kinase activity.

Animals↗
PubMed1989

Sea urchin early and late H4 histone genes bind a specific transcription factor in a stable preinitiation complex.

Early embryonic H4 (EH4) and H2B (EH2B) and late embryonic H4 (LH4) histone genes were transcribed in vitro in a nuclear extract from hatching blastula embryos of the sea urchin Strongylocentrotus purpuratus. The extract was prepared by slight modifications of the methods of Morris et al. (G. F. Morris, D. H. Price, and W. F. Marzluff, Proc. Natl. Acad. Sci. USA 83:3674-3678, 1986) that have been used to obtain a cell-free transcription system from embryos of the sea urchin Lytechinus variegatus. Achievement of maximum levels of transcription of the EH4 and LH4 genes required a 5- to 10-min preincubation of template with extract in the absence of ribonucleoside triphosphates. This preincubation allowed the formation of a stable complex which was preferentially transcribed compared with a second EH4 or LH4 template that was added 10 min later. Although the EH4 gene inhibited both EH4 and LH4 gene transcription in this assay and although the LH4 gene inhibited both EH4 and LH4 genes, neither of these genes inhibited transcription of the EH2B gene. Preincubation with the EH2B gene had no effect on the transcription of subsequently added EH4 or LH4 genes. Using this template commitment assay, we showed that the site of binding of at least one essential factor required for transcription of both EH4 and LH4 genes was located between positions -102 and -436 relative to the 5' terminus of the EH4 mRNA. Moreover, deletion of this region resulted in a reduction in EH4 gene transcription in vitro. The sea urchin gene-specific trans-acting factors, in the analysis of the cis-acting sequences with which they interact, and in biochemical studies on the formation of stable transcription complexes.

Animals↗
PubMed1988

Contractile force of single heart cells compared with muscle strips of frog ventricle.

Single heart cells were obtained from frog ventricle with an enzymatic dispersion technique. Isometric contractile force was measured in these cells by an ultrasensitive force transducer and compared with that generated by multicellular muscle strips under similar conditions. The shape of the single-cell twitch was qualitatively similar to that obtained in intact tissue; however, the time to peak was generally shorter, and the falling phase was prolonged in the single cell compared with the muscle strip. The single-cell contractile force was measured in response to alterations in stimulus rate, resting length, and extracellular Ca2+ concentration [Ca2+]o or by addition of epinephrine; in all cases, the force response resembled the physiological response seen in the muscle strip. However, at a constant stimulation rate, the steady-state amplitude of the single-cell twitch exhibited beat-to-beat variations under all conditions tested, whereas that of the muscle strip was essentially constant. These results may prove to be useful in assessing the suitability of the single-cell preparation as a model for the intact tissue.

Algorithms↗
PubMed1986

An ultrasensitive transducer for measurement of isometric contractile force from single heart cells.

A novel ultrasensitive force transducer suitable for measuring isometric forces generated by single spindle-shaped muscle cells is described. The transducer components are mechanically and electrically simple, consisting of a low power He-Ne laser, a pair of step index, glass optic fibers, and a photodiode detector circuit. To test the operation of the transducer, enzymatically isolated frog ventricular heart cells were used, having peak contractile forces on the order of 100 nN. The transducer presents a number of advantages over existing designs and has a resolution better than 2 nN. As such, it is suitable for excitation-contraction studies in single muscle cells.

Animals↗
PubMed1986

The human type II collagen gene (COL2A1) assigned to 12q14.3.

A cosmid clone containing the entire human type II alpha 1 collagen gene (COL2A1) was used as probe in the Southern analysis of DNA from a panel of human/hamster somatic cell hybrids containing different portions of human chromosome 12. Two of the hybrids exhibited a similar terminal deletion q14.3----qter, but one was positive for the gene while the other was negative. Therefore, the gene must reside in the region q14.3.

Animals↗
PubMed1985

A comparative electrophysiological study of enzymatically isolated single cells and strips of frog ventricle.

Single heart cells were obtained from frog ventricle using an enzymatic dispersion technique. The whole cell variation of the patch clamp technique was used to monitor action potential and cell membrane currents. The clamp circuit could be switched electronically between voltage and current clamp modes. The effects of seal leakage currents were to depolarize the cell, reduce the amplitude of the plateau, and lengthen the action potential duration. A scheme to compensate for these currents is presented. The membrane currents obtained from the single cell under voltage clamp conditions were compared to those obtained from multicellular preparations using the single sucrose gap technique. Hyperpolarizing clamps showed time-dependent, depletion-related K+ currents for the multicellular preparation, whereas for the single cell no such currents were observed. The absence of extracellular accumulation or depletion of K+ in the single cell was confirmed by the lack of post-clamp afterpotentials or changes in resting potential following a train of frequently elicited action potentials. The TTX-insensitive inward current was relatively faster in the single cell, compared to that measured in the multicellular preparation. A delayed time-dependent outward current was observed in the positive potential range for both single and multicellular preparations. The isochronal current-voltage (I-V) relations obtained at 400 ms were N-shaped for both preparations, but was more negative for the single cell at potentials positive to -20 mV. The results indicate a strong similarity between membrane currents obtained in single and multicellular preparations. The differences in the currents in the two preparations are due in large part to accumulation or depletion of K+ in the extracellular space.

Action Potentials↗
PubMed1985

Effect of RP51 gene dosage alterations on ribosome synthesis in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae ribosomal protein rp51 is encoded by two interchangeable genes, RP51A and RP51B. We altered the RP51 gene dose by creating deletions of the RP51A or RP51B genes or both. Deletions of both genes led to spore inviability, indicating that rp51 is an essential ribosomal protein. From single deletion studies in haploid cells, we concluded that there was no intergenic dosage compensation at the level of mRNA abundance or mRNA utilization (translational efficiency), although phenotypic analysis had previously indicated a small compensation effect on growth rate. Similarly, deletions in diploid strains indicated that no strong mechanisms exist for intragenic dosage compensation; in all cases, a decreased dose of RP51 genes was characterized by a slow growth phenotype. A decreased dose of RP51 genes also led to insufficient amounts of 40S ribosomal subunits, as evidenced by a dramatic accumulation of excess 60S ribosomal subunits. We conclude that inhibition of 40S synthesis had little or no effect on the synthesis of the 60S subunit components. Addition of extra copies of rp51 genes led to extra rp51 protein synthesis. The additional rp51 protein was rapidly degraded. We propose that rp51 and perhaps many ribosomal proteins are normally oversynthesized, but the unassembled excess is degraded, and that the apparent compensation seen in haploids, i.e., the fact that the growth rate of mutant strains is less depressed than the actual reduction in mRNA, is a consequence of this excess which is spared from proteolysis under this circumstance.

Diploidy↗
PubMed1983

The rna2 mutation of yeast affects the processing of actin mRNA as well as ribosomal protein mRNAs.

The temperature sensitive rna2 mutation of Saccharomyces cerevisiae causes a rapid and dramatic decrease in the abundance of most ribosomal protein mRNAs We and others have recently shown that the processing of ribosomal protein mRNAs is defective at the nonpermissive temperature, suggesting that inefficient mRNA processing might be responsible for the decline in ribosomal protein mRNA levels. Actin is the only known intron-containing non-ribosomal protein yeast nuclear gene We show here that the processing of actin mRNA is also defective at the nonpermissive temperature in rna2-containing strains. The observation supports the notion that all intron-containing genes are affected in a similar fashion by the rna2 mutation.

Actins↗
PubMed1983

Voltage- and frequency-dependent block of diltiazem on the slow inward current and generation of tension in frog ventricular muscle.

The effects of a new "Ca2+ -antagonist", diltiazem, were studied in frog ventricular myocardium. The single sucrose gap voltage clamp technique was used to control membrane potential and measure membrane current and tension. Diltiazem had no effect on the resting potential, but reduced the slow inward current (Isi) and increased the net outward current. Membrane conductance measurements suggest that the increase in the total membrane current may be due to the suppression of a maintained inward current. The blocking action of diltiazem is frequency-dependent such that at higher frequencies of stimulation, the steady-state amplitude of twitch is reduced. The diltiazem-induced suppression of tension and Isi could be partially reversed by hyperpolarizing the surface membrane for brief intervals. These results suggest that diltiazem blocks Isi in a voltage- and time-dependent manner. These effects of diltiazem on Isi seem to be, in part, responsible for the tension-suppressant effect of the drug.

Action Potentials↗
PubMed1982

Ionic events responsible for the cardiac resting and action potential.

The cardiac action potential is distinguished from other excitatory phenomena by a prominent plateau and by the latent pacemaking capability of cardiac muscle. A review of experimental data suggests that ionic fluxes through gated membrane channels are the primary determinants of the shape of the cardiac action potential. The rapid depolarization phase of the action potential is mediated in part by an ionic channel that resembles the sodium ion (Na+) channel of nerve. A slower channel capable of carrying both calcium ion (Ca2+) and Na+ currents (Isi) also contributes to the upstroke of the action potential. The Ca2+ current through this channel is partly responsible for maintaining the plateau phase of the action potential. Moreover, because this slower channel activates at more positive potentials in partly depolarized myocardium or in specialized conduction tissue such as the sinoatrial and atrioventricular nodes. Two distinct transport systems appear to be the principal regulators of potassium ion (K+) in myocardium. An inwardly rectifying, voltage-dependent K+ channel apparently maintains all K+ conductance at rest and at all potentials negative to --20 mV. A second channel, which is both voltage- and time-dependent, evidently mediates K+ flux during the plateau phase of the action potential. This K+ current activates at potentials positive to --20 mV. The role of coupled transport mechanisms is now well established. The low intracellular concentrations of Na+ and Ca2+ prevailing in the myocardium are maintained by an electrogenic Na+ pump and a Na+-Ca2+ counter-transport system. Current data on carrier-mediated transport systems are insufficient to delineate the role of such mechanisms in the control of cardiac action potential. Further studies are required to provide details of the voltage, temporal and ionic dependence of gated channels, as well as of ionic counter-transport carriers, so that a quantitative reconstruction of cardiac action potential may be attempted.

Animals↗
PubMed1978

[Swine liver ultrastructure following hypothermia and hyperbaric oxygenation preservation].

The livers of 20 pigs were preserved for up to 16 hours, applying hypothermia (4 degrees C) and hyperbaric oxygenation (3 ata). As a preparation solvent we used in one group the solution by Lie (17) which is rich in potassium and glucose, and in the other group the cryoprecipitated serum by Belzer (5). After 8 and 16 hours of preservation, the fine structure was examined light- and electronmicroscopically. After 8 hours of preservation, there were only little reversible changes in both groups. After 16 hours, both groups showed distinct changes of the endoplasmatic reticulum e. g. desorganisation, vesicular dilatation, and dissociation of the mural ribosomes. In addition, there were swellings of mitochondriae and Kupffer's cells. These changes were particularly distinct after preservation in cryoprecipitated serum. In this group a cellular depletion of glucogen was noticeable.

Animals↗