Differential utilization of calcitonin gene regulatory DNA sequences in cultured lines of medullary thyroid carcinoma and small-cell lung carcinoma.

Regulation of expression of the human calcitonin gene was found to differ between two tumor lines of different tissue origin, medullary thyroid carcinoma (TT line) and small-cell lung carcinoma (DMS53 line). Distal 5' DNA elements between -750 and -2000 exhibited a stronger basal activity in DMS53 than in TT cells, whereas proximal DNA sequences between -132 and -252 mediated a dramatic cyclic AMP response in TT but not DMS53 cells.     

Ion selectivity of temperature-induced and electric field induced pores in dipalmitoylphosphatidylcholine vesicles

Temperature and electric field are known to alter the permeability of the bilayer membrane in phospholipid vesicles. A study of cation selectivity of these membrane pores is reported for multilamellar liposomes (MLV) and unilamellar large vesicles (ULV, 95 +/- 5 nm diameter) of dipalmitoylphosphatidylcholine (DPPC). The permeability of ULV to Rb+ was 1.0 X 10(-6) micrograms/s at 22 degrees C and increased to 1.1 X 10(-5) micrograms/s at the gel to liquid-crystalline transition temperature (Tm) of the bilayer, at 42 degrees C. The permeability of ULV to Rb+ continued to increase beyond the Tm and reached 1.0 X 10(-4) micrograms/s at 56 degrees C, a 100-fold increase over the permeability at 22 degrees C. In contrast, the permeability of ULV to Na+ showed a local maximum of 6.0 X 10(-6) micrograms/s at 42 degrees C and decreased at temperatures higher or lower than the Tm. For MLV, the permeability to both Rb+ and Na+ peaked dramatically at the phase transition temperature, 42 degrees C, and subsided at lower and higher temperatures. When ULV were exposed to an electric field, the permeability to Rb+, Na+, and sucrose surged at a field strength of 30 kV/cm; 30 kV/cm can induce a transmembrane potential of 210 mV. In ULV, the electrically perforated lipid bilayer exhibited selectivity for Rb+ over Na+ only at a narrow electric field range, between 31 and 33 kV/cm. For MLV, no well-defined breakdown voltage was recorded.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

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)     

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.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational relaxations of urea- and guanidine hydrochloride-unfolded ferricytochrome c.

Several recent studies of protein the unfolded proteins. In urea- and guanidine HCl-unfolded ferricytochrome c (horse heart), an acid-induced spin state transformation of the heme group has been detected by the heme absorptions, Trp-59 fluorescence, and the intrinsic viscosity of protein. Kinetics of this second conformational transition, by the temperature jump and stopped flow methods, are complex. One rapid reaction (tau1), pH-independent, occurs in a 50-mus range; the second reaction (tau2), in a 1-ms range, depends linearly upon pH and is faster at the alkaline side; a third reaction (tau3), in a 1-s range, shows a sigmoidal transition at pH 5.1 and is faster at the acidic side. The results are consistent with a kinetic scheme which involves protein conformational changes in the transformation of the heme coordination state. The kinetics, along with previous equilibrium studies, indicate that ligand or charge interactions within a protein molecule are not completely prohibited even in strongly denaturing conditions, such as in high concentrations of urea and guanidine HCl. Thus, local structures of peptide chain associated with these interactions can exist in the unfolded protein.     

Slow equilibration of a denatured protein: comparison of the cluster model with the proline isomerization model

The slow equilibration of the denatured state after rapid unfolding of a globular protein is examined by the cluster model of protein folding (Kanehisa &; Tsong, 1978). The detection of this process in ribonuclease A and its acid catalysis have been considered evidence for the proline isomerization model. Our calculation shows that similar kinetic behavior is also expected for the cluster model.     

Enthalpic and entropic contributions to actin stability: calorimetry, circular dichroism, and fluorescence study and effects of calcium

The delta H associated with the thermal unfolding of G-actin has been determined by differential scanning calorimetry (DSC) to be 142 +/- 5 kcal/mol, with the Tm (melting temperature) at 57.2 +/- 0.5 degrees C, at pH 8.0 (heating rate 0.5 K/min). The transition is broad and cannot be treated as a single transition that mimics a two-state process, suggesting the existence of domains. Deconvolution is done to fit it into two quasi-independent two-state transitions. For F-actin, the transition is more cooperative, with a cooperative ratio (the ratio of van't Hoff enthalpy and calorimetric enthalpy) of 1.4, indicating intermonomer interaction. The delta H of the thermal unfolding of F-actin is 162 +/- 10 kcal/mol with a Tm at 67.0 +/- 0.5 degrees C. A state of G-actin similar to that of the heat-denatured form, designated D-actin, is obtained by removing tightly bound Ca2+ with EGTA. The DSC-detectable cooperative transition is completely lost when the free calcium concentration of the medium is 1 x 10(-11) M or lower, using a Ca2+/EGTA buffer system. However, circular dichroism (CD) shows that the helix content of actin, 32% in the G-form, is only partially reduced to 19% in this apo form. The CD spectrum and the helix content of the calcium-depleted actin are almost identical with those of the heat-denatured D form. This loss of 40% of the native helical content is irreversible in both cases. The remaining 60% of the native helical content cannot be further eliminated by heating to 95 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective production of hybridoma cells: antigenic-based pre-selection of B lymphocytes for electrofusion with myeloma cells

Methods for pre-selecting B lymphocytes were studied and investigated. First, biotinylated antigen was used for selecting B lymphocytes. These pre-selected B lymphocytes were then combined with biotinylated myeloma cells by adding streptavidin. The final formula of the selected B cell-myeloma cell was as follows: B cell-(antigen-biotin-strept-avidin-biotin)-myeloma cell. Then, this B cell-myeloma cell conjugate was fused by the pulsed electric field (PEF) method, which fused only those conjugated cells. The fusion efficiency obtained by this method was 3-15-times higher than that obtained by the non-specific poly(ethylene glycol) (PEG) fusion method. Second, avidin-antigen conjugate was used to select B lymphocytes. For this purpose, bifunctional cross-linkers such as N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and m-maleimidobenzoyl N-hydroxysuccinimide (MBS) were chosen. Each reagent contains two heterofunctional groups which can make covalent bond with both Lys and Cys residues. Typical avidin-antigen conjugate is expressed as avidin-SPDP (or MBS)-antigen. Thus, final B cell-myeloma cell conjugate was B cell-antigen-SPDP (or MBS)-avidin-biotin-myeloma cell. The yield of this procedure was of the order of 10(-2). Here, we suggest that the pre-selection of B lymphocytes by biotinylated antigen or avidin-antigen conjugate is a new method of obtaining selected hybridoma cells which produce specific monoclonal antibodies against the antigen used for selecting B lymphocytes.