An improved vaseline gap voltage clamp for skeletal muscle fibers. (1/44)

A Vaseline gap potentiometric recording and voltage clamp method is developed for frog skeletal muscle fibers. The method is based on the Frankenhaeuser-Dodge voltage clamp for myelinated nerve with modifications to improve the frequency response, to compensate for external series resistance, and to compensate for the complex impedance of the current-passing pathway. Fragments of single muscle fibers are plucked from the semitendinosus muscle and mounted while depolarized by a solution like CsF. After Vaseline seals are formed between fluid pools, the fiber ends are cut once again, the central region is rinsed with Ringer solution, and the feedback amplifiers are turned on. Errors in the potential and current records are assessed by direct measurements with microelectrodes. The passive properties of the preparation are simulated by the "disk" equivalent circuit for the transverse tubular system and the derived parameters are similar to previous measurements with microelectrodes. Action potentials at 5 degrees C are long because of the absence of delayed rectification. Their shape is approximately simulated by solving the disk model with sodium permeability in the surface and tubular membranes. Voltage clamp currents consist primarily of capacity currents and sodium currents. The peak inward sodium current density at 5 degrees C is 3.7 mA/cm2. At 5 degrees C the sodium currents are smoothly graded with increasing depolarization and free of notches suggesting good control of the surface membrane. At higher temperatures a small, late extra inward current appears for small depolarizations that has the properties expected for excitation in the transverse tubular system. Comparison of recorded currents with simulations shows that while the transverse tubular system has regenerative sodium currents, they are too small to make important errors in the total current recorded at the surface under voltage clamp at low temperature. The tubules are definitely not under voltage clamp control.  (+info)

The precision of a special purpose analog computer in clinical cardiac output determination. (2/44)

Three hundred dye-dilution curves taken during our first year of clinical experience with the Waters CO-4 cardiac output computer were analyzed to estimate the errors involved in its use. Provided that calibration is accurate and 5.0 mg of dye are injected for each curve, then the percentage standard deviation of measurement using this computer is about 8.7%. Included in this are the errors inherent in the computer, errors due to baseline drift, errors in the injection of dye and acutal variation of cardiac output over a series of successive determinations. The size of this error is comparable to that involved in manual calculation. The mean value of five successive curves will be within 10% of the real value in 99 cases out of 100. Advances in methodology and equipment are discussed which make calibration simpler and more accurate, and which should also improve the quality of computer determination. A list of suggestions is given to minimize the errors involved in the clinical use of this equipment.  (+info)

Computer-controlled automation of radioimmunoassay based on gel entrapment of antibody. (3/44)

We describe a new, broadly applicable approach to radioimmunoassay. Solid-phase binding reagent is prepared by entrapping antibody in polyacrylamide gel. This gel is then fragmented, sieved, and dehydrated in 95 percent ethanol. Upon evaporation of the ethanol, the dry antibody-gel particles are dispensed into miniature disposable plastic columns. A unidirectional flowing stream configuration is used to construct a standard curve for the polypeptide hormone, angiotensin I, used here to exemplify the technique. Good reproducibility of this standard curve is demonstrated. The analytical system includes a computer-controlled sample turntable, digital pipet, and liquid-switching assembly.  (+info)

On-line identification of sensory systems using pseudorandom binary noise perturbations. (4/44)

A technique of on-line identification of linear system characteristics from sensory systems with spike train or analog voltage outputs was developed and applied to the semicircular canal. A pseudorandom binary white noise input was cross-correlated with the system's output to produce estimates of linear system unit impulse responses (UIRs), which were then corrected for response errors of the input transducers. The effects of variability in the system response characteristics and sensitivity were studied by employing the technique with known linear analog circuits. First-order unit afferent responses from the guitarfish horizontal semicircular canal were cross-correlated with white noise rotational acceleration inputs to produce non-parametric UIR models. In addition, the UIRs were fitted by nonlinear regression to truncated exponential series to produce parametric models in the form of low-order linear system equations. The experimental responses to the white noise input were then compared with those predicted from the UIR models linear convolution, and the differences were expressed as a percent mean-square-error (%MSE). The average difference found from a population of 62 semicircular canal afferents was relatively low mean and standard deviation of 10.2 +/- 5.9 SD%MSE, respectively. This suggests that relatively accurate inferences can be made concerning the physiology of the semicircular canal from the linear characteristics of afferent responses.  (+info)

Coagulation-time determination with automatic multivariable analysis, by use of a miniature centrifugal fast analyzer. (5/44)

Use of a miniature Centrifugal Fast Analyzer for the parallel photometric monitoring of the coagulation process is shown to have a number of advantages. These include a choice of optical modes, virtually simultaneous initiation and observation of the coagulation process for a number of patient-plasma samples and an on-board control sample, small sample and reagent volume requirements, and automatic determination of a number of diagnostically useful variables (including relative fibrinogen content) from the data recorded in a single run. Also, the system is shown to give results that correlate well with those obtained by conventional techniques for determination of prothrombin time.  (+info)

Influence of an aggregated multienzyme system on transient time: kinetic evidence for compartmentation by an aromatic-amino-acid synthesizing complex of Neurospora crassa. (6/44)

The aromatic complex of Neurospora crassa is an aggregated multienzyme system which catalyzes five consecutive reactions in the central pathway leading to the biosynthesis of the aromatic amino acids. In an attempt to understand the physiological importance of this complex in particular, as well as the importance of cellular organization of enzyme systems in general, we have isolated the complex and have begun to characterize its catalytic properties. Optimum conditions for the assay of the overall 5-step reaction catalyzed by the partially purified complex have been determined. An analog computer was programmed to represent an unaggregated system of five enzymes with rate constants identical to those found for the constituent enzymes of the complex. By direct comparison, it was shown that the lags (transient times) obtained for the overall reaction were 10-15 times longer for the hypothetical unaggregated system than for the complex. We conclude from these data that the aggregated multienzyme system compartmentalizes intermediate substrates during the course of the overall reaction. We suggest that, in addition to "channeling" intermediates of competing pathways, reduction of the transient time may be an important consequence of the containment of intermediates within a physically associated enzyme sequence. The fact that the aromatic complex exhibits a second catalytic property unique to aggregated enzyme systems, "coordinate activation" [Welch, G.R. & Gaertner, F.H. (1975) Arch. Biochem. Biophys., in press] indicates that the physical association of these enzymes may have more than one physiological function.  (+info)

The dissociation of flavin coenzymes from trypsin-solubilized NADPH/Cytochrome c (P-450) reductase of pig-liver microsomes. (7/44)

The change in fluorescence emission at 520 nm after excitation at 365 nm was used to investigate the effect of pH and ionic strength on the dissociation of flavin cofactors from microsomal NADPH/cytochrome c (P-450) reductase. In the unmodified enzyme both the FAD and FMN moieties appeared to dissociate at a similar rate and followed first-order kinetics. The rate constant for the dissociation was increased by low pH and high ionic strength, particularly in the range pH 4.4-3.8 (0.02 M acetate buffer) where the rate constants increased 80-fold. Modification of the enzyme by treatment with p-chloromercuribenzoate enhanced the rate of flavin dissociation and, in the region of pH 4, resulted in a biphasic increase in fluorescence consistent with two simultaneous parallel first-order dissociations. It was concluded that p-chloromercuribenzoate treatment modified the protein so that the two flavin cofactors dissociated at different rates. Using the measured rate constants for the dissociations, and the known variation in fluorescence of flavin nucleotides with pH, an analogue computer simulation of the dissociation as well as a manual curve-fitting procedure showed that the biphasic response could be explained as a simultaneous rapid dissociation of FAD and a slower loss of FMN from the protein.  (+info)

A NEW INTERPRETATION OF THE DYNAMIC CHANGES OF THE POTASSIUM CONDUCTANCE IN THE SQUID GIANT AXON. (8/44)

The solutions, n(t), of the differential equation dn/dt = alpha (1 - n) n (4 - 6n + 4n(2) - n(3)) - betan(2) (4 - 6n + 4n(2) - n(3)) in which alpha and beta are instantaneous functions of membrane potential, are shown to fit with good accuracy the time courses of the rise of potassium conductance during depolarizing steps in clamp potential, found experimentally by Hodgkin and Huxley and by Cole and Moore. The equation is derived by analysing the dynamic behaviour of a system consisting of a square array of interacting pores. The possible role of Ca(++) ions in this system is discussed.  (+info)