Search PubMed⌕ Search

Biomedical subjects

T Y Tsong

Publications and source records attributed to T Y Tsong.

At least 19 recordsLinked to original sources
PubMed2001

Targeting antigen-specific receptors on B lymphocytes to generate high yields of specific monoclonal antibodies directed against biologically active lower antigenic peptides within presenilin 1.

We describe a targeting technique that selects antigen-specific receptors on B lymphocytes using antigen driven selective production of monoclonal antibodies which are directed against functional peptide sequences within the presenilin 1 molecule that is believed to be related to the early-onset of familial Alzheimer's disease. Three different peptide sequences of presenilin 1 were constructed, one including the region around the amino acid position 300, where the putative cleavage site exists and the other two present in the N- and C-terminal regions of that site. The efficiency in production of the desired monoclonal antibodies was at least 5-40-fold that obtained with the poly(ethylene glycol) (PEG)-mediated method. In addition, monoclonal antibodies directed against each of the peptide sequence displayed a high specificity for the corresponding peptide, in contrast to the lack of success using the PEG method. Also, the selection of surface immunoglobulin receptors on B lymphocytes by the peptides of interest was confirmed by immunofluorescent analysis. Here we demonstrate that targeting B lymphocytes results in the successful and efficient production of highly specific monoclonal antibodies against the lower antigenic peptide sequences.

Animals↗
PubMed1999

Biological effects of electric shock and heat denaturation and oxidation of molecules, membranes, and cellular functions.

Direct exposure of cells in suspension to intense electric pulses is known to produce damages to cell membranes and supramolecular organizations of cells, and denaturation of macromolecules, much like injuries and tears seen in electric trauma patients. Thus, the system has been used as a laboratory model for investigating the biochemical basis of electric injury. An intense electric pulse can produce two major effects on cells--one caused by the field, or the electric potential, and the other by current, or the electric energy. The field-induced transmembrane potential can produce electro-conformational changes of ion channels and ion pumps and, when the potential exceeds the dielectric strength of the cell membrane (approximately 500 mV for a pulse width of a few ms), electro-conformational damages and electroporations of membrane proteins and lipid bilayers. These events lead to passage of electric current through the membrane-porated cells and to heating of cell membranes and cytoplasmic contents. The subsequent denaturation of cell membranes and cytoplasmic macromolecules brings about many complex biochemical reactions, including oxidation of proteins and lipids. The combined effects may cripple the cells beyond repair. This communication will focus on the thermal effects of electric shock. After a brief review of the current state of knowledge on thermal denaturation of soluble enzymes and muscle proteins, this paper will describe experiments on the thermal denaturation of cellular components and functions, such as nucleosomes, and the electron transport chain and ATP synthetic enzymes of the mitochondrial inner membranes. Data will show that lipid peroxidation and the subsequent loss of the energy-transducing ability of the cells may occur even at moderate temperatures between 40 degrees C and 45 degrees C. However, lipid peroxidation may be prevented with reducing reagents such as mercaptoethanol, dithiothreitol, and ascorbic acid. Reactivation of denatured cellular proteins and functions may also be possible and a strategy for doing so is discussed.

Animals↗
PubMed1997

Fluctuation-driven directional flow in biochemical cycle: further study of electric activation of Na,K pumps.

Directional flow of information and energies is characteristic of many types of biochemical reactions, for instance, ion transport, energy coupling during ATP synthesis, and muscle contraction. Can a fluctuating force field, or a noise, induce such a directional flux? Previous work has shown that Na,K-ATPase of human erythrocyte can absorb free energy from an externally applied random-telegraph-noise (RTN) electric field to pump Rb+ up its concentration gradient. However, the RTN field used in these experiments was constant in amplitude and would not mimic fluctuating electric fields of a cell membrane. Here we show that electric fields which fluctuate both in life time and in amplitude, and thus, better mimicking the transmembrane electric fields of a cell, can also induce Rb+ pumping by Na,K-ATPase. A Gaussian-RTN-electric field, or a field with amplitude fluctuating according to the Gaussian distribution, with varied standard deviation (sigma), induced active pumping of Rb+ in human erythrocyte, which was completely inhibited by ouabain. Increased values for sigma led to a nonmonotonic reduction in pumping efficiency. A general formula for calculating the ion transport in a biochemical cycle induced by fluctuating electric field has been derived and applied to a simple four-state electroconformational coupling (ECC) model. It was found that the calculated efficiency in the energy coupling decreased with increasing sigma value, and this effect was relatively small and monotonic, whereas experimental data were more complex: monotonic under certain sets of conditions but nonmonotonic under different sets. The agreement in general features but disagreement in some fine features suggest that there are other properties of the electric activation process for Na,K-ATPase that cannot be adequately described by the simple ECC model, and further refinement of the ECC model is required.

Biophysical Phenomena↗
PubMed1996

Least activation path for protein folding: investigation of staphylococcal nuclease folding by stopped-flow circular dichroism.

Is the pathway of protein folding determined by the relative stability of folding intermediates, or by the relative height of the activation barriers leading to these intermediates? This is a fundamental question for resolving the Levinthal paradox, which stated that protein folding by a random search mechanism would require a time too long to be plausible. To answer this question, we have studied the guanidinium chloride (GdmCl)-induced folding/unfolding of staphylococcal nuclease [(SNase, formerly EC 3.1.4.7; now called microbial nuclease or endonuclease, EC 3.1.31.1] by stopped-flow circular dichroism (CD) and differential scanning microcalorimetry (DSC). The data show that while the equilibrium transition is a quasi-two-state process, kinetics in the 2-ms to 500-s time range are triphasic. Data support the sequential mechanism for SNase folding: U3 <--> U2 <--> U1 <--> N0, where U1, U2, and U3 are substates of the unfolded protein and N0 is the native state. Analysis of the relative population of the U1, U2, and U3 species in 2.0 M GdmCl gives delta-G values for the U3 --> U2 reaction of +0.1 kcal/mol and for the U2 --> U1 reaction of -0.49 kcal/mol. The delta-G value for the U1 --> N0 reaction is calculated to be -4.5 kcal/mol from DSC data. The activation energy, enthalpy, and entropy for each kinetic step are also determined. These results allow us to make the following four conclusions. (i) Although the U1, U2, and U3 states are nearly isoenergetic, no random walk occurs among them during the folding. The pathway of folding is unique and sequential. In other words, the relative stability of the folding intermediates does not dictate the folding pathway. Instead, the folding is a descent toward the global free-energy minimum of the native state via the least activation path in the vast energy landscape. Barrier avoidance leads the way, and barrier height limits the rate. Thus, the Levinthal paradox is not applicable to the protein-folding problem. (ii) The main folding reaction (U1 --> N0), in which the peptide chain acquires most of its free energy (via van der Waals' contacts, hydrogen bonding, and electrostatic interactions), is a highly concerted process. These energy-acquiring events take place in a single kinetic phase. (iii) U1 appears to be a compact unfolded species; the rate of conversion of U2 to U1 depends on the viscosity of solution. (iv) All four relaxation times reported here depend on GdmCl concentrations: it is likely that none involve the cis/trans isomerization of prolines. Finally, a mechanism is presented in which formation of sheet-like chain conformations and a hydrophobic condensation event precede the main-chain folding reaction.

Circular Dichroism↗
PubMed1995

Cell fission and formation of mini cell bodies by high frequency alternating electric field.

We report the use of high frequency alternating electric fields (AC) to induce deformation of sea urchin eggs, leading to budding of membrane vesicles or fission of cells. Several mini cell bodies can be prepared from a single egg by carefully manipulating the frequency and amplitude of the AC field and the ratio between the interelectrode spacing and the cell diameter, alpha. alpha values between 2.2 and 3.5 have been found to be optimal for inducing fission of sea urchin eggs. In a typical experiment, a sea urchin egg (diameter = 75 microns), suspended in a low ionic medium (conductance < 2 mS/m), was located under the microscope between two platinum wire electrodes, separated by a distance of approximately 200 microns. A medium strength AC field (< 100 V/cm at 2 MHz) was applied to attract the egg to one of the two electrodes via dielectrophoresis. This process took place in a few seconds. The voltage was then slowly increased to approximately 1000 V/cm over approximately 30 s. The cell elongated and separated into two fragments, the larger one containing the nucleus. When the field was turned off, the mother cell and the daughter vesicle retracted to form spherical mini cell bodies that appear to be stable as assessed by the absence of swelling for the duration of the experiment (approximately 15 min). This indicates that membranes of these mini cell bodies were not leaky to ions and small molecules. This procedure could be repeated a few times to make several mini cell bodies from a single egg. With practice, several minicell bodies could also be prepared in a single fission experiment by adjusting the field parameters and the a value. Cell fission is a result of the mechanical stress produced by the AC field. These procedures may be used to prepare mini membrane vesicles for voltage clamp experiments or to perform microsurgical manipulation of cells, embryos, or chromosomes.

Animals↗
PubMed1994

Chain condensation in protein folding.

Three reactions (relaxation times: tau 1 = 140 +/- 8 ms, tau 2 = 840 +/- 30 ms and tau 3 = 30 + 3 s, at pH 7.0 at 25 degrees C) have been resolved by the stopped-flow kinetic method for the folding of the acid denatured staphylococcal nuclease (Chen et al (1991) J Mol Biol 220, 771; Biochemistry (1992) 31, 1483). Of the three reactions only the tau 2 reaction was dependent on the viscosity of the solution in a manner consistent with the diffusion/collision/coalescence model. Experiments with site-directed mutagenesis suggest that the forming of local structures by the electrostatic interactions between Glu75 and His121 and Lys9 may induce the chain condensation.

Diffusion↗
PubMed1994

Recognition and processing of randomly fluctuating electric signals by Na,K-ATPase.

Previous work has shown that Na,K-ATPase of human erythrocytes can extract free energy from sinusoidal electric fields to pump cations up their respective concentration gradients. Because regularly oscillating waveform is not a feature of the transmembrane electric potential of cells, questions have been raised whether these observed effects are biologically relevant. Here we show that a random-telegraph fluctuating electric field (RTF) consisting of alternating square electric pulses with random lifetimes can also stimulate the Rb(+)-pumping mode of the Na,K-ATPase. The net RTF-stimulated, ouabain-sensitive Rb+ pumping was monitored with 86Rb+. The tracer-measured, Rb+ influx exhibited frequency and amplitude dependencies that peaked at the mean frequency of 1.0 kHz and amplitude of 20 V/cm. At 4 degrees C, the maximal pumping activity under these optimal conditions was 28 Rb+/RBC-hr, which is approximately 50% higher than that obtained with the sinusoidal electric field. These findings indicate that Na,K-ATPase can recognize an electric signal, either regularly oscillatory or randomly fluctuating, for energy coupling, with high fidelity. The use of RTF for activation also allowed a quantitative theoretical analysis of kinetics of a membrane transport model of any complexity according to the theory of electroconformational coupling (ECC) by the diagram methods. A four-state ECC model was shown to produce the amplitude and the frequency windows of the Rb(+)-pumping if the free energy of interaction of the transporter with the membrane potential was to include a nonlinear quadratic term. Kinetic constants for the ECC model have been derived. These results indicate that the ECC is a plausible mechanism for the recognition and processing of electric signals by proteins of the cell membrane.

Biophysical Phenomena↗
PubMed1994

Comparison of kinetics of formation of helices and hydrophobic core during the folding of staphylococcal nuclease from acid.

Our previous kinetic study of the acid and base-induced folding/unfolding transitions of staphylococcal nuclease (SNase) has monitored Trp-140 fluorescence. Trp-140 is located near the flexible COOH terminus and whether or not its fluorescence reflects the overall conformation of the protein has yet to be established. Here we show that the fluorescence intensity of Try-140 correlated closely with the thermal stability (i.e., the calorimetric enthalpy, delta Hcal, of unfolding) of the protein in the pH range 7 to 2.5, confirming that it is a good measure of the overall protein structure. Circular dichroism (CD) at 222 nm, which reflects the helical content of the protein molecule, was used to follow the same folding/unfolding transition in order to compare kinetics of the helix formation and of the appearance of the hydrophobic core. In addition to the three kinetic phases reported earlier with the fluorescence detection, there were a rapid reaction (completed within the 25 ms mixing time of the instrument), which comprised 15% of the signal, and a very slow reaction (time constant > 300 s), which comprised 19% of the signal. With the fluorescence detection for the folding from acid, only 5% of the signal occurred in the rapid phase and there was no reaction slower than 300 s. By comparing kinetics of folding at pH 7 by the CD and fluorescence detection methods, we concluded that: (a) Roughly 15% of the helix content of SNase accumulated before significant changes in the hydrophobic environment (< 5%) of Trp-140 could be detected. The rapid appearance of CD signal reminiscent of helix formation within 25ms would be consistent with the framework model of protein folding. Note, however, that, 15% of the 22% helix content of the protein amounts to an equivalent of fewer than 5 amino acid residues. (b) For the time-resolved signal between 2 ms and 300s, kinetics measured by both properties are consistent with the sequential model, D4 = D3 = D2 = D1 = No (the four Ds are the four substates of the denatured protein and No is the native state). The major folding step by both signals is the D1 to No transition, which gave approximately a 50% change in fluorescence and CD and had a time constant of 160 ms at 25 degree C, pH7.0. (c) The slow phase with the CD signal (>300 s), which is insensitive to Trp-140 fluorescence, has been assigned to be the cis/trans isomerization of Pro-1 17 by other studies. (d) Kinetics in the unfolding direction are consistent with the above interpretation.

Amino Acid Sequence↗
PubMed1994

On the efficiency and reversibility of active ligand transport induced by alternating rectangular electric pulses.

The stationary-state kinetic properties of a simplified two-state electro-conformational coupling model (ECC) in the presence of alternating rectangular electric potential pulses are derived analytically. Analytic expressions for the transport flux, the rate of electric energy dissipation, and the efficiency of the transducing system are obtained as a function of the amplitude and frequency of the oscillation. These formulas clarify some fundamental concept of the ECC model and are directly applicable to the interpretation and design of experiments. Based on these formulas, the reversibility and the degree of coupling of the system can be studied quantitatively. It is found that the oscillation-induced free energy transduction is reversible and tight-coupled only when the amplitude of the oscillating electric field is infinitely large. In general, the coupling is not tight when the amplitude of the electric field is finite. Furthermore, depending on the kinetic parameters of the model, there may exist a "critical" electric field amplitude, below which free energy transduction is not reversible. That is, energy may be transduced from the electric to the chemical, but not from the chemical to the electric.

Carrier Proteins↗
PubMed1993

Study of mechanisms of electric field-induced DNA transfection. V. Effects of DNA topology on surface binding, cell uptake, expression, and integration into host chromosomes of DNA in the mammalian cell.

Neumann and coworkers (Neumann, E., M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider. 1982. EMBO J. 1:841-845) have shown that the efficiency of pulsed electric field (PEF)-induced DNA transfection of mouse L-cells by the thymidine kinase gene is several times higher for the linear DNA than for the closed circular DNA. Transfection of Escherichia coli bacteria by several plasmids indicates that the transfection efficiency was much higher for the closed circular/supercoiled (sc-) and circular/relaxed (cr-) DNA than for the linearized (In-) DNA (Xie, T. D., L. Sun, H. G. Zhao, J. A. Fuchs, and T. Y. Tsong. 1992. Biophys. J. 63:1026-1031). To resolve these conflicting observations, we have systematically examined electrotransfection of NIH3T3 mouse fibroblast by the plasmids, pRSVcat, pRSVneo, and pRSVgpt. Mg(2+)-facilitated surface binding of DNA before, and DNA uptake by 3T3 cells after treatment with PEF, were monitored by 3H-labeled plasmids. Transfection efficiency was evaluated by both the transient expression of chloramphenicol acetyltransferase (cat) activity 2-3 days after, and the permanent expression of neomycin phosphotransferase (neo) and xanthine-guanine phosphoribosyltransferase (gpt) genes in the transformants 2 weeks after the PEF treatment. Our results indicate that cell surface binding and PEF-induced cell uptake of DNA did not depend on the topology of DNA. However, both the transient and the permanent expression of the plasmids were three to five times more efficient for the cr-DNA and the sc-DNA than for the in-DNA. These results indicate that electrotransfection of cells involves several steps: the cation-dependent binding of DNA to the cell surface, the electric field-driven DNA entry into the cells, the transient expression of DNA, and the integration of DNA into the host chromosomes. For understanding mechanisms of electrotransfection, only the DNA binding to the cell surface and the electric field assisted membrane-crossing of DNA are relevant. Both the expression of the loaded DNA and the DNA integration into the host chromosomes depend more on the properties of the cell and its interactions with a foreign gene. Since these properties and interactions will be similar irrespective of the method chosen to facilitate DNA transfer, they are not relevant for the study of mechanisms of electrotransfection. Our results also support the idea that the PEF-induced cellular uptake of DNA is mainly by the electrophoresis of the surface bound DNA across the plasma membrane.

3T3 Cells↗
PubMed1992

Kinetic evidence of microscopic states in protein folding.

Staphylococcal nuclease unfolds at acidic pHs and refolds at neutral pH. Previous kinetic analysis based on both the direct pH jump and the sequential pH jump, from a native condition (pH 7.0) to pHs beyond unfolding transition zones (pH 3.0 and pH 12), and vice versa, supports the mechanism, D3<-->D2<-->D1<-->N0, in which N0 is the native state and D's are the three substates of the denatured form [Chen, H.M., You, J.L., Markin, V.S., & Tsong, T.Y. (1990) J. Mol. Biol. 220, 771-778; Chen, H.M., Markin, V.S., & Tsong, T.Y. (1992) Biochemistry 31, 1483-1491]. Here we show that both the single- and the double-pH jump kinetics of folding and unfolding to the intermediate pHs (3.4-5.0, i.e., in the transition zone), in which both the native and the denatured states coexist, are not compatible with this simple sequential model. At 25 degrees C, log tau 1(-1) (for the D1<-->N0 step) and log tau 2(-1) (for the D2<-->D1 step) vs pH show a square root of-shaped dependence on the final pH, with minimal values (tau 1(-1) of 0.56 s-1 and tau 2(-1) of around pH 3.9. The third relaxation tau 3 (for the D3<-->D2 step, 35 s) was independent of pH in the range 3.4-8.5. The square root of-shaped dependence on pH of log tau 1(-1) and log tau 2(-1) cannot be reproduced by the above but can be accounted for if each of N0, D1, and D2 is composed of many microscopic states in rapid equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen-Ion Concentration↗
PubMed1992

Molecular recognition and processing of periodic signals in cells: study of activation of membrane ATPases by alternating electric fields.

A molecule which is immobilized, oriented or tumbling more slowly than the frequency of a periodic field, may interact with the field to produce chemical effects that are uncommon in a homogeneous solution. Among these effects are the alteration of the rate of a chemical reaction and the exchange of energy between the oscillating field and the conformation of the molecule. When certain conditions are satisfied, this exchange allows the molecule to absorb and couple the energy of the field to drive an endergonic reaction. The efficiency of energy coupling depends on field strength and frequency and on the ligand concentration. There are windows of these parameters to achieve efficient coupling. These windows can be expressed in terms of the rate constants and equilibrium constants of the catalytic reactions, and the amplitude and frequency of the periodic field. This mechanism allows cells to receive, process and transmit energy of high and medium level periodic potentials by means of membrane enzymes or receptors. A theory for the transduction of electric energy, electroconformational coupling (ECC) will be discussed. The electric field induced cation pumping activities of Na,K-ATPase and Ca-ATPase of human erythrocytes and the ATP synthetic activity of beef heart mitochondrial ATPase will then be used to test an ECC membrane transport model. For the processing of low level periodic signals, a theory of an oscillatory activation barrier (OAB), which considers resonance transduction between an oscillating field and the activation barrier of the rate limiting step in an enzymic reaction, will be discussed. The OAB mechanism successfully interprets the AC stimulated ATP hydrolysis activity of Ecto-ATPase from chicken oviduct and F0F1-ATPase from beef heart. We propose that mechanisms similar to an OAB model are adopted by cells to sense weak electric, acoustic, mechanical, concentration (i.e., chemical potential) and other types of signals, and to communicate with other cells by these signals. The experimental data and mechanistic information presented in this communication give us a glimpse of the molecular electronic designs in living cells. This information is also relevant with respect to environmental issues. Environmental electromagnetic fields and sonic pollutants may interfere with normal communications of cells and organisms. Their benefit, if any, and detrimental effects can be assessed and dealt with only if we fully understand mechanisms of cellular interactions with these fields and pollutants, at the molecular level.

Adenosine Triphosphatases↗
PubMed1992

pH-induced folding/unfolding of staphylococcal nuclease: determination of kinetic parameters by the sequential-jump method.

On the basis of previous stopped-flow pH-jump experiments, we have proposed that the acid- and alkaline-induced folding/unfolding transition of staphylococcal nuclease, in the time range 2 ms to 300 s, follows the pathway N0 in equilibrium with D1 in equilibrium with D2 in equilibrium with D3, in which D1, D2, and D3 are three substates of the unfolded state and N0 is the native state. The stopped-flow "double-jump" technique has been employed to test this mechanism and to determine the rate constants which would not be accessible by the direct pH jump because of the lack of fluorescence signal, i.e., the rates for the conversion of D1 to D2 and of D2 to D3. In the forward jump, a protein solution kept at pH 7.0 was mixed with an acidic or alkaline solution to the final pH of 3.0 or 12.2, respectively. The mixed solution was kept for varying periods of time, called the delay time, tD. A second mixing (the back jump) was launched to bring the protein solution back to pH 7.0. The time course of the Trp-140 fluorescence signals recovered in the back jump was analyzed as a function of tD. Kinetics of the unfolding were found to be triphasic by the double-jump method, contrary to the monophasic kinetics observed by the direct pH jump. Complex kinetics of unfolding are expected with the proposed kinetic scheme.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme Stability↗