Comparison of MPEG-1 digital videotape with digitized sVHS videotape for quantitative echocardiographic measurements.

Digital format is rapidly emerging as a preferred method for displaying and retrieving echocardiographic studies. The qualitative diagnostic accuracy of Moving Pictures Experts Group (MPEG-1) compressed digital echocardiographic studies has been previously reported. The goals of the present study were to compare quantitative measurements derived from MPEG-1 recordings with the super-VHS (sVHS) videotape clinical standard. Six reviewers performed blinded measurements from still-frame images selected from 20 echocardiographic studies that were simultaneously acquired in sVHS and MPEG-1 formats. Measurements were obtainable in 1401 (95%) of 1486 MPEG-1 variables compared with 1356 (91%) of 1486 sVHS variables (P <.001). Excellent agreement existed between MPEG-1 and sVHS 2-dimensional linear measurements (r = 0.97; MPEG-1 = 0.95[sVHS] + 1.1 mm; P <.001; Delta = 9% +/- 10%), 2-dimensional area measurements (r = 0.89), color jet areas (r = 0.87, p <.001), and Doppler velocities (r = 0.92, p <.001). Interobserver variability was similar for both sVHS and MPEG-1 readings. Our results indicate that quantitative off-line measurements from MPEG-1 digitized echocardiographic studies are feasible and comparable to those obtained from sVHS.

Evaluation of phase transition errors in heat capacity calorimeters using SPICE simulated RC models.

A technique is presented which allows the development of extremely complex (> 5000 components) RC models of calorimeters and provides simulation under a wide variety of inputs. A commercially available circuit simulation program (SPICE) is used to 'build' RC models of relaxation and scanning calorimeter designs used to measure heat capacity. The instrument models are constructed using subcircuits to represent small elements of the materials used in the designs. Only simple linear equations are needed to evaluate the RC values in the subcircuit. The subcircuits also use voltage controlled switches to simulate phase transitions where lambda, C, and delta h can change value instantaneously at predetermined temperatures. The resulting simulations of the two designs provide an ability to predict instrument sensitivity to lambda and C independently. Simulated outputs agree with measured outputs within 10%. Model simulations show serious errors in the heat capacity determinations from both designs during phase transitions. Interactions between lambda, C, and delta h are shown for both designs. The technique provides a means to construct, evaluate, and optimize a calorimeter design completely in software.

An iterative method for the deconvolution of microcalorimeter thermograms.

An iterative method for the deconvolution of microcalorimetry thermograms suitable for small digital computers is presented. The method employs a measured impulse response function directly as the deconvolution kernel, thus explicit system simulation is not required. Data are presented showing the performance of the method and the exchange of signal-to-noise ratio for time resolution that is made when deconvolution techniques are employed. An improvement in the system time resolution of fifty times is demonstrated with measured data.

A computer-controlled all-tantalum stopped-flow microcalorimeter with microjoule resolution.

A new all-tantalum differential stopped-flow heat-conduction microcalorimeter with microjoule resolution has been developed. The instrument consists of two matched channels, each of which has two reagent inlet lines. A computer is used to process the data and control the syringe drive system which runs the samples through the calorimeter. The reagents are mixed in 0.6 s in ratios of 1:1, 1:2, 1:2.5, 1:4, 1:5, or 1:10. The priming volume from the loading port to the mixer is 1 ml and the reaction volume of the detection tube is 160 microliters. The instrument has a sensitivity of 1.60 J/V.s and a differential baseline stability of 100 nJ/s (p-p) over a 4 h period. The sample size can be reduced to 27 microliters with only a 12% loss in sensitivity. With an electrical step power input, the 10-90% response is 40 s. By using a data decomposition scheme, the response time can be improved to 1 s which allows the direct measurement of moderately fast reaction kinetics. With water/water mixes, differential heats of mixing are typically (+/-) 2 microJ with a standard deviation of (+/-) 2.5 microJ. Reaction heats in the 20-50 microJ range can be measured with a standard deviation of (+/-) 3 microJ. A fast reaction, e.g. HCl dilution, can be completed in 150 s. When loading and priming times are included, 25 reactions can be completed in 120 min. A chilled water jacket is used to allow operation over a temperature range of 4 degrees C to 50 degrees C.

A differential heat-conduction microcalorimeter for heat-capacity measurements of fluids.

A heat-conduction calorimeter has been developed for measuring small changes in heat capacity of milligram samples of membrane lipid dispersed in water as a function of temperature. The operation of the instrument is based on the principle that the thermal response of the sample to a short (10 s), electrically generated heat burst is a function of the diffusivity of the sample. Modeling studies of the instrument's performance have revealed that the output response after the heat burst is a function of only the heat capacity, rho Cp. Calibration of the instrument experimentally confirmed this behavior. This feature obviated the need to measure the thermal conductivity in order to determine rho Cp from the diffusivity equation, eta = lambda/rho Cp. The calorimeter has the following characteristics: reproducibility of loading: +/- 400 microJ/C degrees.cm3; baseline stability: +/- 10 microJ/C degrees.cm3 per 36 h; resolution (+/- 1 S.D.): +/- 50 microJ/C degrees.cm3; sample size 600 microliters.

An optimized differential heat conduction solution microcalorimeter for thermal kinetic measurements.

Heat conduction calorimeters are widely used in the biological sciences, but baseline instability, low resolution, electrical noise and motion artifacts have limited their utility. Two main sources of noise, baseline fluctuation or drift and a motion artifact, were traced to amplifier drift, a small (0.015 degrees C) gradient within the constant temperature cylinder, and the method of installing the thermopiles. The addition of heaters to the top and bottom of the cylinder reduced the gradient to approximately 0.003 degrees C and greatly reduced the slow component of the motion artifact. The drift error was reduced by proper mounting of the amplifier and its external components and the enclosure of the calorimeter in a temperature-controlled box. An R-C model of the heat flow in the calorimeter was developed which was employed to discover several means of increasing sensitivity without increasing the rise-time of the calorimeter. Analysis, also based on the model, showed that variations in the air gap between the cell and cell holder can be a major source of error when the calorimeter is used to investigate the kinetics of a chemical reaction. This analysis also showed that the time for the heat to flow through the solution in the cell can be the dominant factor in determining the rise-time of the instrument. The heat conduction calorimeter described here has improved characteristics: a baseline stability of 200 nJ x s-1 (peak-to-peak) over a 48 h period; a resolution of 200 nJ x s-1; a sensitivity of 6.504 +/- 0.045 J x V-1 x s-1 referred to the sensor output; and a rise-time of 122 s for the 10-90% response.

A stopped-flow mixer device for a batch microcalorimeter application to NAD-NADase reaction.

A new molded polypropylene, diamond-like carbon (DLC)-coated mixing cell has been developed for use in the batch microcalorimeter. Reagent volume can be varied from 25 microliters to 100 microliters. A 10 microcalorie reaction heat can be measured to 5%. Repeat reactions can be done as often as every 10 min for a fast reaction. Reactions can be started within 1 h or less after loading. A pre-equilibrator and a temperature-controlled syringe drive unit permit solutions to be stored at 4 degrees C while being run at any temperature from -20 degrees C to 40 degrees C. The kinetics and enthalpy of reaction of NAD-NADase have been measured. delta H is about 21 kcal/mol endothermic.

Thermodynamics of Cro protein-DNA interactions.

Using a highly sensitive pulsed-flow microcalorimeter, we have measured the changes in enthalpy and determined the thermodynamic parameters delta H, delta S degree, delta G degree, and delta C(p) for Cro protein-DNA association reactions. The reactions studied include sequence-nonspecific DNA association and sequence-specific DNA associations involving single- and multiple-base alterations and/or single-amino acid alteration mutants. (i) The association of Cro protein with nonspecific DNA at 15 degrees C is characterized by delta H = +4.4 kcal.mol-1 (1 cal = 4.18J), delta S degrees = 49 cal.mol-1.K-1, delta G degrees = -9.7 kcal.mol-1, and delta Cp congruent to 0; the association with specific high-affinity operator OR3 DNA is characterized by delta H = +0.8 kcal.mol-1, delta S degree = 59 cal.mol-1.K-1, delta G degree = -16.1 kcal.mol-1, and delta Cp = -360 cal.mol-1.K-1, respectively. Both nonspecific and specific Cro-DNA associations are entropy-driven. (ii) Plots of delta H vs. delta Cp and delta S degree vs. delta Cp for the 20 association reactions studied fall into two correlation groups with linear slopes of +9.4 K and -20.5 K and of -0.03 and -0.14, respectively. These regression lines have common intercepts, at the delta H and delta S degree values of nonspecific association (where delta Cp congruent to 0). The results suggest that there are, at least, two distinct conformational subclasses in specific Cro-DNA complexes, stabilized by different combinations of enthalpic and entropic contributions. The delta G degree and delta Cp values form an approximately single linear correlation group as a consequence of compensatory contributions from delta H and delta S degree to delta G degree and to delta Cp. Cro protein-DNA associations share some similar thermodynamic properties with protein folding, but their overall energetics are quite different. Although the nonspecific complex is stabilized predominantly by electrostatic forces, it appears that H bonds, van der Waals contacts, hydrophobic effects, and charge interactions all contribute to the stability (delta G degree and delta Cp) of the specific complex. (iii) The variations in the values of the thermodynamic parameters are in general accord with our knowledge of the structure of the Cro-DNA complex.

Thermodynamic characterization of daunomycin-DNA interactions: comparison of complete binding profiles for a series of DNA host duplexes.

Using a combination of spectroscopic and calorimetric techniques, we have determined complete thermodynamic binding profiles (delta G degree, delta H degree, and delta S degree) for the complexation of daunomycin to a series of 10 polymeric DNA duplexes. We find the resulting drug binding data to be sensitive to the base composition and sequence of the host duplex, with the binding free energies ranging from -7.5 to -10.8 kcal/mol of bound drug and the binding enthalpies ranging from +4.11 to -10.76 kcal/mol of bound drug at 25 degrees C. The smaller range in the free energy term reflects the impact of large enthalpy-entropy compensations. We observe that the three synthetic duplexes which exhibit the highest daunomycin binding affinities all contain GC (or IC) base pairs as part of alternating purine/pyrimidine sequence motifs, with these high binding affinities being strongly enthalpy driven at 25 degrees C. Specific comparisons between the binding profiles for daunomycin complexation with select pairs of host duplexes lead to the following observations: (1) The presence or absence of a major-groove methyl group does not alter daunomycin binding thermodynamics. (2) The presence or absence of a minor-groove amino group does alter daunomycin binding thermodynamics. (3) Duplexes with different base compositions but identical minor-groove functionality exhibit similar daunomycin binding thermodynamics. (4) Homopolymeric duplexes composed of either AT or AU base pairs, but not GC base pairs, exhibit large enthalpy-entropy compensations in their daunomycin binding profiles. We propose interpretations of these and other features of our thermodynamic data in terms of specific daunomycin-DNA interactions deduced from available structural data.

Thermodynamic characterization of daunomycin-DNA interactions: microcalorimetric measurements of daunomycin-DNA binding enthalpies.

We report the first direct determination of binding enthalpies for the complexation of monomeric daunomycin with a series of 10 polymeric DNA duplexes. These measurements were accomplished by using a recently developed stopped-flow microcalorimeter capable of detecting reaction heats on the microjoule level. This enhanced sensitivity allowed us to measure daunomycin-DNA binding enthalpies at monomeric drug concentrations (e.g., 10-20 microM), thereby precluding the need to correct for daunomycin self-association, as has been required in previous batch calorimetric studies [Remeta, D. P., Marky, L. A., & Breslauer, K. J. (1984) Abstracts of Pittsburgh Conference and Exposition on Analytical Chemistry and Applied Spectroscopy, 838a; Breslauer, K. J., Remeta, D. P., Chou, W. Y., Ferrante, R., Curry, J., Zaunczkowski, D., Snyder, J. G., & Marky, L. A. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8922-8926]. We correct the published daunomycin-DNA binding enthalpies measured by batch calorimetry at higher drug concentrations (e.g., 0.5-1.0 M) for the enthalpy contribution associated with the binding-induced disruption of drug aggregates. The requisite correction term was obtained from a van't Hoff analysis of temperature-dependent NMR measurements on daunomycin solutions. We find remarkable agreement between the net binding enthalpies derived from these corrected batch calorimetric data and the corresponding binding enthalpies measured directly by stopped-flow microcalorimetry. The enhanced sensitivity of the stopped-flow instrument also allowed us to evaluate the influence of drug binding density on the daunomycin-DNA binding enthalpies. This assessment was accomplished by conducting stopped-flow calorimetric measurements over a range of seven different drug-to-phosphate ratios (r). For most of the 10 DNA host duplexes studied, we find that the daunomycin binding enthalpies exhibit small but significant r dependencies. The sensitivity of the stopped-flow instrument also enabled us to detect significant dilution enthalpies for several of the drug-free DNA duplexes, a quantity generally assumed to be negligible in previous studies. We discuss the binding enthalpies, their dependence on binding density, and the duplex dilution enthalpies in terms of the influence of base composition, sequence, conformation/hydration, and binding cooperativity on the sign and the magnitudes of the daunomycin-DNA binding enthalpy data reported here.

Critical temperature for unilamellar vesicle formation in dimyristoylphosphatidylcholine dispersions from specific heat measurements.

Using a heat conduction calorimeter with very high resolution (+/- 0.00005 J/degrees C.cm3), we have measured the specific heat CpL between 25 and 35 degrees C of dimyristoylphosphatidylcholine (DMPC) in aqueous dispersions. Previous studies of the temperature dependence of the chemical potential of DMPC in the L alpha phase (lamellar, liquid crystalline) indicated that a dispersion consisting only of unilamellar vesicles forms spontaneously at a critical temperature T* of 29.0 degrees C. Our present measurements show an anomaly in CpL between 28.70 and 29.50 degrees C: the curve for CpL versus T first decreases and then exhibits an inflection point at 28.96 degrees C before it flattens. This anomaly is attributed to the transformation from multilamellar dispersion to unilamellar vesicles at T* = 28.96 degrees C. Two independent properties of the CpL data also indicate T* is a critical point for the formation of unilamellar vesicles: (a) the time to reach equilibrium upon changing temperature increased dramatically between 28.7 and 28.96 degrees C, increasing as (T* - T)-1; at T > T* the dramatic "slowing-down" phenomenon was not observed. This slowing-down near T* is a general characteristic of critical phenomena. (b) The free energy change for the multilamellar-unilamellar transformation was obtained from the CpL-T data over this temperature interval and found to be 3.2 J/mol or 0.016 ergs/cm2 of bilayer, in agreement with other estimates of the interaction energy between neutral bilayers. We conclude with a discussion of the implications for membrane bilayer stability of these newly identified dynamic properties of the transformation.