Lowry protein assay using an automatic microtiter plate spectrophotometer

The method of protein determination reported by Lowry et al. (1951, J. Biol. Chem. 193, 265-275) has been adapted for use with 96-well microtiter plates and an automatic microplate spectrophotometer. The spectrophotometer has been interfaced with a computer which plots the standard curve and calculates the protein content of each sample. The adapted method offers advantages over previously reported methods in that it is more rapid and uses a smaller sample volume (100 microliters) for samples containing 3-300 micrograms/ml (0.3-30 micrograms/assay) of protein. The method of Bensadoun and Weinstein (1976, Anal. Biochem. 70, 241-252) for precipitating microgram amounts of protein away from substances which interfere with the Lowry assay has also been adapted to this microplate procedure. These techniques should be particularly useful for laboratories where large numbers of samples containing a wide range of protein concentrations are assayed.     

Decay of inspiratory muscle pressure during expiration in anesthetized cats

In six spontaneously breathing anesthetized cats (pentobarbital sodium, 35 mg/kg) we studied the antagonistic pressure developed by the inspiratory muscles during expiration (PmusI). This was accomplished in two ways: 1) with our previously reported method (J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 52: 1266-1271, 1982) based on the measurement of changes in lung volume and airflow during spontaneous expiration, together with determination of the total passive respiratory system elastance and resistance; and 2) measurement of the time course of changes in tracheal/pressure after airway occlusion at end inspiration, up to the moment when the inspiratory muscles become completely relaxed. The agreement between the two methods is generally good, both in the amplitude of PmusI and in its time course. We also applied the first method to spontaneous expirations through added linear resistive loads. These did not alter the relative decay of PmusI. Thus in anesthetized cats the braking action of the inspiratory muscles does not decrease when expiratory resistive loads are added, i.e., when such braking is clearly not required.     

"Creep currents" in single frog atrial cells may be generated by electrogenic Na/Ca exchange

The objective of these experiments was to test the hypothesis that the "creep currents" induced by Na loading of single frog atrial cells (Hume, J. R., and A. Uehara. 1986. Journal of General Physiology. 87:833) may be generated by an electrogenic Na/Ca exchanger. Creep currents induced by Na loading were examined over a wide range of membrane potentials. During depolarizing voltage-clamp pulses, outward creep currents were observed, followed by inward creep currents upon the return to the holding potential. During hyperpolarizing voltage-clamp pulses, creep currents of the opposite polarity were observed: inward creep currents were observed during the pulses, followed by outward creep currents upon the return to the holding potential. The current-voltage relations for inward and outward creep currents in response to depolarizing or hyperpolarizing voltage displacements away from the holding potential all intersect the voltage axis at a common potential, which indicates that inward and outward creep currents may have a common reversal potential under equilibrium conditions and may therefore be generated by a common mechanism. Measurements of inward creep currents confirm that voltage displacements away from the holding potential rapidly alter equilibrium conditions. Current-voltage relationships of inward creep currents after depolarizing voltage-clamp pulses are extremely labile and depend critically upon the amplitude and duration of outward creep currents elicited during preceding voltage-clamp pulses. An optical monitor of mechanical activity in single cells revealed (a) a similar voltage dependence for the outward creep currents induced by Na loading and tonic contraction, and (b) a close correlation between the time course of the decay of the inward creep current and the time course of mechanical relaxation. A mathematical model of electrogenic Na/Ca exchange (Mullins, L.J. 1979. Federation Proceedings. 35:2583; Noble, D. 1986. Cardiac Muscle. 171-200) can adequately account for many of the properties of creep currents. It is concluded that creep currents in single frog atrial cells may be attributed to the operation of an electrogenic Na/Ca exchange mechanism.     

Paradoxical loss of excitation with high intensity pulses during electric field stimulation of single cardiac cells

Transmembrane potential responses of single cardiac cells stimulated at rest were studied with uniform rectangular field pulses having durations of 0.5-10 ms. Cells were enzymatically isolated from guinea pig ventricles, stained with voltage sensitive dye di-8-ANEPPS, and stimulated along their long axes. Fluorescence signals were recorded with spatial resolution of 17 microm for up to 11 sites along the cell. With 5 and 10 ms pulses, all cells (n = 10) fired an action potential over a broad range of field amplitudes (approximately 3-65 V/cm). With 0.5 and 1 ms pulses, all cells (n = 7) fired an action potential for field amplitudes ranging from the threshold value (approximately 4-8 V/cm) to 50-60 V/cm. However, when the field amplitude was further increased, five of seven cells failed to fire an action potential. We postulated that this paradoxical loss of excitation for higher amplitude field pulses is the result of nonuniform polarization of the cell membrane under conditions of electric field stimulation, and a counterbalancing interplay between sodium current and inwardly rectifying potassium current with increasing field strength. This hypothesis was verified using computer simulations of a field-stimulated guinea pig ventricular cell. In conclusion, we show that for stimulation with short-duration pulses, cells can be excited for fields ranging between a low amplitude excitation threshold and a high amplitude threshold above which the excitation is suppressed. These results can have implications for the mechanistic understanding of defibrillation outcome, especially in the setting of diseased myocardium.     

Alternative interpretation for the osmotic response of human erythrocytes

In a recent publication, Heubusch et al. (J Cell. Physiol, 122:266-272, 1985) reported changes of erythrocyte volume measured by the Coulter counter technique over a wide range of osmolalities (160 to 3000 m0sm). Their results showed a partially hindered, nonlinear response, in contrast to classical observations made over more restricted osmolality ranges, using other methods. The authors suggested the underlying cause of this behavior to be a mechanical resistance of the membrane cytoskeleton. In this paper, we wish to offer a different interpretation of their results on erythrocyte osmotic behavior, based on similar experiments carried out in our laboratory, and supported by previous analyses from the literature. In particular, it is shown that the shape-factor correction to the electronic sizing measurement can correctly account for the observed deviations from linearity in the hypotonic range. In contrast, increased chemical nonideality and eventual hemolysis are the likely factors responsible for the behavior in the hypertonic range.     

The effects of pH on force and stiffness development in mouse muscles

Changes in force and stiffness during contractions of mouse extensor digitorum longus and soleus muscles were measured over a range of extracellular pH from 6.4 to 7.4. Muscle stiffness was measured using small amplitude (less than 0.1% of muscle length), high frequency (1.5 kHz) oscillations in length. Twitch force was not significantly affected by changes in pH, but the peak force during repetitive stimulation (2, 3, and 20 pulses) was decreased significantly as the pH was reduced. Changes in muscle stiffness with pH were in the same direction, but smaller in extent. If the number of attached cross-bridges in the muscle can be determined from the measurement of small amplitude, high frequency muscle stiffness, then these findings suggest that (a) the number of cross-bridges between thick and thin filaments declines in low pH and (b) the average force per cross-bridge also declines in low pH. The decline in force per cross-bridge could arise from a reduction in the ability of cross-bridges to generate force during their state of active force production and (or) in an increased percentage of bonds in a low force, "rigor" state.     

Contrast Enhanced MRI Assessment of Tumor Blood Volume After Application of Electric Pulses

The effect of application of short, intense electric pulses on tumor blood volume was investigated using albumin-(Gd-DTPA)₃₀ contrast-enhanced magnetic resonance imaging (MRI). One of paired SA-1 fibrosarcoma tumors implanted in each flank of A/J mice was treated with electric pulses. MRI was performed dynamically before and after intravenous administration of albumin-(Gd-DTPA)₃₀ (0.02 mmol Gd/kg), and fractional tumor blood volume was estimated. MRI images of tumors exposed to electric pulses showed no enhancement at 30 min after injection of albu-min-(Gd-DTPA)₃₀. However, marked enhancement was observed in paired tumors of the same mice that were not exposed to electric pulses. A significant difference in blood volume was observed between nontreated tumors and tumors treated with electric pulses. Application of electric pulses to the tumors significantly reduced blood volume in the tumors. Therefore, through a reduction in tumor blood volume, electric pulses may, besides producing electroporation of cells, exert antitumor effectiveness by entrapping drugs within the tumors.     

Electroporator with automatic change of electric field direction improves gene electrotransfer <Emphasis Type="Italic">in-vitro</Emphasis>

Background  

Gene electrotransfer is a non-viral method used to transfer genes into living cells by means of high-voltage electric pulses. An exposure of a cell to an adequate amplitude and duration of electric pulses leads to a temporary increase of cell membrane permeability. This phenomenon, termed electroporation or electropermeabilization, allows various otherwise non-permeant molecules, including DNA, to cross the membrane and enter the cell. The aim of our research was to develop and test a new system and protocol that would improve gene electrotransfer by automatic change of electric field direction between electrical pulses.     

Modulation of luteinizing hormone pulse amplitude by the frequency of luteinizing hormone-releasing hormone stimulation and by testosterone in castrated, hypothalamic-lesioned male rats

The frequency of spontaneous luteinizing hormone (LH) pulses is thought to be a direct result of the frequency of luteinizing hormone-releasing hormone (LHRH) pulses from the hypothalamus. By contrast, the amplitude of spontaneous LH pulses may be controlled by several factors other than the amplitude of LHRH pulses. We tested two hypotheses: 1) that LH pulse amplitude is determined in part by the frequency of LHRH pulses of constant magnitude, and 2) that testosterone (T) exerts a direct feedback effect on the pituitary gland to regulate LH pulse amplitude. Gonadal feedback was eliminated by castrating adult male rats (n = 20). Endogenous LHRH secretion was eliminated by lesioning the medial basal hypothalamus. Serum LH levels (0.19 +/- 0.04 ng/ml RP-2, mean +/- SEM) and T levels (0.15 +/- 0.02 ng/ml), measured several weeks after hypothalamic lesioning, confirmed the hypogonadotropic hypogonadal state of the animals. During a 8-h period, unanesthetized, unrestrained animals were injected with 40-ng pulses of LHRH via catheters into the jugular vein, and blood samples for LH measurement were drawn at 10-min intervals. The LHRH pulse interval was 20 min during the first 4 h in all animals. The pulse interval was doubled to 40 min in half of the animals (n = 10) during the next 4 hours; in the other 10 animals, the pulse interval was maintained constant at 20 min throughout the study. Within both of these groups, one-half of the animals (n = 5) were infused with T to achieve a physiological level of T in serum (2.46 +/- 0.36 ng/ml at 4 h), while the other half received vehicle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Automated region of interest analysis of dynamic Ca²⁺ signals in image sequences

Ca(2+) signals are commonly measured using fluorescent Ca(2+) indicators and microscopy techniques, but manual analysis of Ca(2+) measurements is time consuming and subject to bias. Automated region of interest (ROI) detection algorithms have been employed for identification of Ca(2+) signals in one-dimensional line scan images, but currently there is no process to integrate acquisition and analysis of ROIs within two-dimensional time lapse image sequences. Therefore we devised a novel algorithm for rapid ROI identification and measurement based on the analysis of best-fit ellipses assigned to signals within noise-filtered image sequences. This algorithm was implemented as a plugin for ImageJ software (National Institutes of Health, Bethesda, MD). We evaluated the ability of our algorithm to detect synthetic Gaussian signal pulses embedded in background noise. The algorithm placed ROIs very near to the center of a range of signal pulses, resulting in mean signal amplitude measurements of 99.06 ± 4.11% of true amplitude values. As a practical application, we evaluated both agonist-induced Ca(2+) responses in cultured endothelial cell monolayers, and subtle basal endothelial Ca(2+) dynamics in opened artery preparations. Our algorithm enabled comprehensive measurement of individual and localized cellular responses within cultured cell monolayers. It also accurately identified characteristic Ca(2+) transients, or Ca(2+) pulsars, within the endothelium of intact mouse mesenteric arteries and revealed the distribution of this basal Ca(2+) signal modality to be non-Gaussian with respect to amplitude, duration, and spatial spread. We propose that large-scale statistical evaluations made possible by our algorithm will lead to a more efficient and complete characterization of physiologic Ca(2+)-dependent signaling.     

Depolarization-activated potentiation of the T fiber synapse in the blue crab

The blue crab T fiber synapse, associated with the stretch receptor of the swimming leg, has a nonspiking presynaptic element that mediates tonic transmission. This synapse was isolated and a voltage clamp circuit was used to control the membrane potential at the release sites. The dependence of transmitter release on extracellular calcium, [Ca]o, was studied over a range of 2.5-40 mM. A power relationship of 2.7 was obtained between excitatory postsynaptic potential (EPSP) rate of rise and [Ca]o. Brief presynaptic depolarizing steps, 5-10 ms, presented at 0.5 Hz activated EPSP's of constant amplitude. Inserting a 300-ms pulse (conditioning pulse) between these test pulses potentiated the subsequent test EPSPs. This depolarization-activated potentiation (DAP) lasted for 10-20 s and decayed with a single exponential time course. The decay time course remained invariant with test pulse frequencies ranging from 0.11 to 1.1 Hz. The magnitude and decay time course of DAP were independent of the test pulse amplitudes. The magnitude of DAP was a function of conditioning pulse amplitudes. Large conditioning pulses activated large potentiations, whereas the decay time constants were not changed. The DAP is a Ca-dependent process. When the amplitude of conditioning pulses approached the Ca equilibrium potential, the magnitude of potentiation decreased. Repeated application of conditioning pulses, at 2-s intervals, did not produce additional potentiation beyond the level activated by the first conditioning pulse. Comparison of the conditioning EPSP waveforms activated repetitively indicated that potentiation lasted transiently, 100 ms, during a prolonged release. Possible mechanisms of the potentiation are discussed in light of these new findings.     

Circadian rhythms in Neurospora: a new measurement, the reset zone

The authors define a new feature of a circadian rhythm, the reset zone, and point out its usefulness for predictions concerning oscillator behavior. The reset zone measures the responses of a circadian system to resetting pulses. It can be easily determined from a phase transition curve (PTC), which is simply a phase response curve (PRC) replotted as new phase versus old phase (Winfree's format). The reset zone is the range of new phases seen in such a plot and has two potentially useful characteristics: its size and its midpoint. A series of experiments with Neurospora involving temperature pulses indicated that the size of the reset zone changed in a nonlinear way in response to both the duration of 40 degrees C pulses and to the magnitude of temperature change for 3-h pulses. Other existing data are replotted to show how the reset zone size varies with growth temperature and with the period of different clock mutants. Employing exclusively reset zone data within the framework of a limit cycle displacement model, an equation is formulated that predicts the relative changes in the values of state variables of the oscillator for changes in any given environmental condition, such as temperature. Examples are also drawn from other organisms, such as hamsters, Gonyalaux, Kalanchoe, and Drosophila, illustrating the usefulness of the reset zone measurement. It can be used as a numerical scale for assessing the strength of a pulse, for comparing the relative effects of a given pulse applied to different organisms or mutants, for determining the directionality of the changes in state variables produced by various types of pulses, and possibly for measuring clock amplitude.     

Tumor Growth Inhibited by Low-Voltage Amplitude and 5-kHz Frequency Electrochemotherapy

The most important unpleasant sensation of electrochemotherapy is muscle contraction. One of the causes of this discomfort is electrochemotherapy in the low-frequency range (1 Hz). To resolve this problem, there are two solutions: first, increasing the repetition frequency of electric pulses above the tetanic frequency and, second, reducing the voltage amplitude. This study examines the antitumor effectiveness of treatment using low electric fields and high frequency in the presence and absence of chemotherapeutic agents. High-voltage amplitude electrochemotherapy was performed by eight pulses, at 1,000 V/cm, of 100-μs duration at 1-Hz and 5-kHz repetition frequency. In the low-voltage amplitude protocol, 4,000 pulses, of 100-μs duration at 5-kHz repetition frequency with 70, 100 and 150 V/cm were delivered to invasive ductal carcinoma tumors after intratumoral injection of bleomycin. Our data demonstrate significant differences in tumor volumes and the curability rate between mice treated by 70 V/cm compared to other groups. Electrochemotherapy, which is specified by a higher repetition frequency of electric pulses (5 kHz) and low voltage, inhibits tumor growth. This protocol has a comparable effect to 1-Hz pulse repetition electric pulses with high voltage. Based on these results, the 4,000 pulses of 70 V/cm with 5-kHz frequency are most effective. This protocol demonstrates inhibition of tumor growth without any need for drug administration.     

Modeling and Automatic Feedback Control of Tremor: Adaptive Estimation of Deep Brain Stimulation

This paper discusses modeling and automatic feedback control of (postural and rest) tremor for adaptive-control-methodology-based estimation of deep brain stimulation (DBS) parameters. The simplest linear oscillator-based tremor model, between stimulation amplitude and tremor, is investigated by utilizing input-output knowledge. Further, a nonlinear generalization of the oscillator-based tremor model, useful for derivation of a control strategy involving incorporation of parametric-bound knowledge, is provided. Using the Lyapunov method, a robust adaptive output feedback control law, based on measurement of the tremor signal from the fingers of a patient, is formulated to estimate the stimulation amplitude required to control the tremor. By means of the proposed control strategy, an algorithm is developed for estimation of DBS parameters such as amplitude, frequency and pulse width, which provides a framework for development of an automatic clinical device for control of motor symptoms. The DBS parameter estimation results for the proposed control scheme are verified through numerical simulations.     

Noninvasive cardiac output estimation: a preliminary study

 The availability of a simple-to-use, automatic measurement system for noninvasive flow estimation is imperative, given the clinical demand for an acceptable noninvasive procedure rather than the standard invasive procedure of thermodilution. A method for calculating cardiac output from noninvasively derived pressure pulses has been developed, and the results of a preliminary evaluation study on post-cardiac surgery patients for whom invasive flow measures were readily available for comparison are provided in this report. The proposed method relies on fast Fourier transform (FFT) analysis of pulses measured externally at the carotid and femoral pressure points. A transfer function of the aorta is computed from digitally filtered pulse measurements, and a tapered model of the aorta is parametrically adapted using a simplex optimization algorithm so that its transfer function matches that derived experimentally. An aortic input impedance term is obtained from the optimized model and utilized along with the carotid pulse (analogous to input voltage) to compute aortic flow. In addition to its automation, attractive features of this method include the requirement for relatively few pulses for analysis as well as considerable resistance to noise artifact. For 59 data records collected from 54 post-cardiac surgery patients, the average flow measurements computed over several pulses compare well with the standard, invasive method of thermodilution. Preliminary results also indicate a strong potential for tracking changes in cardiac output over time, and invite further use of the method in monitoring hemodynamically unstable patients. Received: 18 February 1997 / Accepted in revised form: 12 May 1997     

The determination of sensitivity parameters for auxin-induced H+-efflux from Avena coleoptile segments

Abstract A method is described for the measurement of auxin-induced H⁺-efflux from small populations of Avena coleoptile segments. The method allows the simultaneous investigation of the kinetics of rapid auxin responses over a wide concentration range. IAA promoted linear rates of H⁺-efflux and the change in amplitude of response occurred mainly over a low, narrow concentration range (10–50 μmol m^-3). The sigmoidal curve of best-fit to each set of dose-response data was determined using non-linear regression techniques, allowing the objective determination of characteristic tissue sensitivity parameters (R_MIN, R_MAX, K_D and p). The sensitivity parameters for the auxin-type herbicide fluroxypyr are also presented as well as IAA parameters determined in the presence of abscisic acid and the ‘antiauxin’ PCIB. The interpretation of the parameter values and the potential use of sensitivity parameter analysis for the evaluation of theories concerning plant hormone action and interaction is discussed.     

Assessment of the Mulder and Van Doorn kinetic procedure and rapid centrifugal analysis of UDP-glucuronosyltransferase activities

The optimal experimental conditions of the enzyme assay described by Mulder and Van Doorn (1975, Biochem J. 151, 131-140) for the measurement of UDP-glucuronosyltransferase activities were tested towards structurally different aglycones. This assessment of this assay revealed that addition of Triton X-100 as enzyme activator was necessary because of its apparent inhibitory effects on interfering reactions. Under these conditions, accordance of the data with results published in the literature was obtained. We present for the first time an UDP-glucuronosyltransferase assay adapted on a fast analyser centrifuge which allows a rapid and sensitive measurement of enzyme activity that is very useful for kinetic constant determination, without consuming a large volume of reagents.     

Restoration of contractions of isolated heart arrested in result of progressing hypothermia with electrical stimuli and no warming up of the heart

In the rat isolated heart, the effect of cooling with the velocity 0.2 degrees C/min and its stimulation with rectangle electrical pulses (19 Hz, 100 ms, 1-5 V amplitude) on the heart mechanical activity in mcWatt/g was studied as the product of heart shortening on contraction strength and heart rate. The heart shortening was measured by the Langendorf method. Heart contractions activated the recording system with the aid of a hook at the apex. The Ringer-Lokk solution served as perfusate pumped with peristaltic pump. After cold arrest of automatic contractions (on the average at 9.3 +/- 0.7 degrees C), the heart continued to respond with contractions to electrical pulses of 5 V amplitude, the power of the heart contractions at 10 degrees C being on the average 35 % of the initial power at 20 degrees C. Excitability and contractility of the heart terminate at 5.3 +/- 0.4 degrees C.     

A sensitive fluorometric assay for the determination of DNA

A sensitive fluorometric assay for DNA determination using m-diaminobenzoic acid dihydrochloride for the reaction with deoxyribose liberated by perchloric acid is described. Fluorescence is proportional to the amount of DNA over the range 0.05–5 μg of DNA when the reaction is conducted in a volume of 400 μl and over the range 0.01–0.5 μg when the reaction is conducted in a volume of 40 μl. The assay is suitable for estimating DNA both in perchloric acid extracts and in complex mixtures with protein and RNA. The determination of DNA in cells grown as a monolayer by the described method is simple and rapid.     

Gradient enhanced selective experiments in the 1H NMR chemical shift assignment of the skeleton and side-chain resonances of stigmasterol, a phytosterol derivative

The applicability of homonuclear gradient enhanced NMR experiments is demonstrated in the structure determination of steroid derivatives using stigmasterol as a model compound. High resolution 1H NMR spectra of steroids very often display well resolved multiplets usually in the low-field region, and these signals can be used as starting points in several selective NMR experiments to study scalar (J coupling), and dipolar (NOE) interactions. Selective excitation was carried out using a double pulsed-field gradient spin-echo sequence (DPFGSE) in which 180 degrees Gaussian pulses are sandwiched between sine shaped z-gradients. Scalar interactions were studied by homonuclear DPFGSE-COSY, DPFGSE-relay-COSY and DPFGSE-TOCSY experiments, while DPFGSE-NOESY was used to monitor spatial environment of the selectively excited proton. These methods provided unambiguous assignments for signals of the main skeleton and the side-chain of the steroid molecule. In addition, they allowed determination of the conformationally important homonuclear proton-proton coupling constants (J).