Effects of buprenorphine on intracerebral collagenase-induced hematoma in Sprague-Dawley rats.

We evaluated the effects of buprenorphine (0.05 mg/kg intraperitoneally) after collagenase-induced intracerebral hemorrhage in Sprague-Dawley rats. Methods of evaluation included serum biochemistry, behavioral tests (neurologic exam and rotarod treadmill), and histopathology. Serum biochemistry parameters showed no change after surgery in controls and buprenorphine-treated animals. At 48 h after collagenase injections, the performance of treated rats on the rotarod treadmill test was not significantly different from that of untreated rats, but the neurologic exams of treated rats showed significantly improved performance. Although the volume of the hematoma was reduced with buprenorphine, the number of necrotic neurons in the penumbra was significantly increased. These data indicate that administration of buprenorphine led to neurologic and histopathologic differences in a rat model of intracerebral hemorrhage, and data from such studies should be interpreted carefully if an opioid analgesic is used to minimize pain.

Decreased levels of myotonic dystrophy protein kinase (DMPK) and delayed differentiation in human myotonic dystrophy myoblasts.

Muscle cell cultures derived from a myotonic dystrophy (DM1) fetus were established in order to determine on the one hand, whether the differentiation of DM1 myoblasts is altered and, on the other hand, whether the levels of myotonic dystrophy protein kinase (DMPK) protein is decreased in DM1 muscle cells. DM1 myoblasts isolated from a quadriceps of a 12-weeks old fetus proliferate at a similar rate as normal myoblasts isolated from a quadriceps of an unaffected 15-weeks old fetus but their maturation is altered as shown by the decreased levels in slow myosin heavy chain protein. In contrast, no change was observed in the expression of vimentin, myogenin and embryonic myosin heavy chain. The levels of DMPK transcripts sharply increased during myoblast differentiation and the mutant DMPK transcripts are retained in discrete foci in the nuclei of muscle cells. The levels of 85-kDa DMPK protein was reduced by about 50% in DM1 cells compared with normal cells. Our study demonstrates that delay in DM1 myoblast maturation is associated with nuclear retention of mutant DMPK transcripts and decreased levels of DMPK protein.

Epithelial and stromal uterine cells cultured in vitro protect bovine sperm from hydrogen peroxide.

It is known that large amounts of leukocytes colonize the uterus, and that these leukocytes can produce considerable quantities of hydrogen peroxide (H2O2) and other reactive oxygen species that are toxic to sperm. It has been shown recently that oviductal fluid has a catalase that helps to maintain sperm motility. Therefore, the current experiment was performed to determine if a similar mechanism of protection exists against peroxides within uterine cells. Sperm motility and velocity were recorded after a 6h incubation in 1) conditioned media in the presence of endometrial cells, 2) conditioned media without endometrial cells, 3) control media (48h without cells) over endometrial cells, or 4) control media alone. All these treatments were performed in the presence or absence of added catalase. Conditioned media, endometrial cells and catalase had a significant positive effect on the maintenance of sperm motility and velocity. Addition of anti-catalase antibodies did not neutralize the beneficial effect of the conditioned media. However, the concentrations of aromatic amino acids, known substrates for sperm amino acid oxidase, were significantly lower in uterine conditioned media as compared to control medium. This reduction of aromatic amino acids was in correlation with reduced H2O2 production by sperm as estimated by chemiluminescence. These results suggest that epithelial and stromal uterine cells do not maintain sperm motility by secreting catalase in the conditioned media, but rather by reducing the levels of aromatic amino acids and thus of peroxides generated in the presence of spermatozoa.

Modeling a non-inactivating delayed rectifier cardiac current using voltage clamp data.

This paper describes a new parameter estimation method applicable to experimental voltage-clamp records. The method is based on the Hodgkin-Huxley (HH) representation of a generic non-inactivating delayed rectifier current (IK) which can be assimilated to the delayed rectifier potassium current of cardiac cells. The model involves a single gating variable of activation (chi) of degree (lambda chi). Its parameters include the voltage-dependent steady-state characteristic (chi infinity), time constant tau chi, the degree lambda chi as a positive integer, and the maximal conductance gK. The method is based on linear optimization. It implements a series of least-squares minimization steps to calculate a first estimate of each model parameter, followed by global minimization to obtain final estimates. The required data, in the form of ionic current responses, correspond to standard voltage-clamp protocols. The effects of noise are minimized by avoiding the use of the time derivative of IK in the calculations. Simulated voltage-clamp data using either a HH model or a five-state Markov chain (MC) model served two purposes: (i) to test the performance of the HH parameter estimation method, and (ii) to study the suitability of the HH model to reproduce data generated by models other than HH. A nominal MC model was obtained by fitting its current responses to those of the HH model. Rate constants of the nominal MC model were then modified and voltage-clamp current responses were generated. Excellent results were obtained with HH and nominal MC data. Data sets generated by a 20% change in the rate constants of the nominal MC model showed that the closed-state rate constants have only a limited influence on the HH parameter estimates, whereas changes in the closed-to-open rate constants produce substantial effects. Nevertheless, a given MC data set can be fitted quite closely by a HH model. In the light of these simulation results it is indicated that an hybrid HH-MC representation of IK data would be more flexible than a straight HH model by removing some of the constraints between the rate constants, and less cumbersome than a straight MC model by substantially reducing the number of parameters to be estimated.

Estimation of the steady-state characteristics of the Hodgkin-Huxley model from voltage-clamp data.

In a companion paper in this issue we show that all the parameters and functions of the Hodgkin-Huxley (HH) model can be calculated in a unique and optimal manner from voltage-clamp peak current data when the steady-state activation (x infinite) and inactivation (z infinite) characteristics are known. Assuming that x infinite and z infinite can be adequately expressed by a Boltzmann equation with two parameters, the present paper describes an optimization procedure to estimate these parameters from peak current data without any constraint on the time constants of activation and inactivation. The required voltage-clamp data are the peak ionic current value (Ip) and its time of occurrence (tp), as provided by two complementary voltage-clamp protocols involving, in each case, a single fixed value of clamp potential. The performance of the procedure was very good with simulated medium- or high-resolution data as it was then possible to determine with confidence the degrees of the gating variables. The performance was also very good with low-resolution data, provided that the degrees of the gating variables were chosen correctly. Good results were also obtained in the presence of Gaussian noise. On the other hand, estimates of x infinite and z infinite based on normalization of peak current measurements always give uncertain results that are likely to be incorrect in a number of circumstances. It is concluded that the HH model can be a useful tool for the interpretation of voltage-clamp peak current data when a reasonable database is available.

Sodium-calcium exchange: derivation of a state diagram and rate constants from experimental data.

A mechanism is developed for Na(+)-Ca2+ exchange using a new approach made possible by the availability of computer software that allows the systematic search of a large parameter space for optimum sets of parameters to fit multiple sets of experimental data. The approach was to make the experimental data dictate the form of the mechanism: the qualitative features of the data dictating the number and nature of the states of the exchanger and their interrelationship, and the quantitative aspects of the data dictating the values of the rate constants that govern the amount of each state relative to the total amount of exchanger. A single set of experimental data served this initial purpose, namely, observations of equilibrium Ca(2+)-Ca2+ exchange in cardiac sarcolemmal vesicles (Slaughter et al., 1983, J. biol. Chem. 258, 3183-3190). From this data a minimum mechanism was induced having 56 states (SYM56), which gave satisfactory quantitative fits to the experimental data. With this set of parameters additional experimental data were fitted, from the same preparation, the single cardiac cell and the squid giant axon, with some changes in parameters, but none dramatic. In spite of the symmetric nature of the mechanism, i.e. binding constants for Na+ and Ca2+ do not depend on the orientation of the binding sites, the mechanism exhibits marked asymmetric behavior similar to that observed experimentally. Finally, in accounting for Ca(2+)-Ca2+ exchange in the absence of monovalent cations, Ca2+ influx becomes dependent on intracellular Ca(2+)--an unexpected outcome--exactly in keeping with the "essential activator" role of intracellular Ca2+ observed by DiPolo & Beaugé (1987, J. gen. Physiol. 90, 505-525). Observations of Na(+)-Ca2+ exchange in the retinal rod outer segment are well fitted with a simplified version of SYM56 comprising 25 states (namely, SYM25), supporting the notion that the exchanger in the retinal rod outer segment differs from that in cardiac sarcolemma and squid axon. Maximum turnover rate of 840 sec-1 for SYM56 and 20 sec-1 for SYM25 are comparable to those reported for the exchanger in cardiac muscle and retinal rod outer segment, respectively.

Modeling the dynamic features of the electrogenic Na,K pump of cardiac cells.

The purpose of this paper is to examine the dynamic features of the electrogenic Na,K pump of cardiac cells, based on a comparative analysis of a mechanistic model and an ad hoc mathematical description of the Na,K pump. Both representations are incorporated into a modified version of the Beeler-Reuter model for the ventricular membrane, and the resulting action potential models are studied under conditions of repetitive stimulation at steady rates between 0 and 3 Hz. The two Na,K pump representations have nearly identical steady-state characteristics of sensitivity to internal Na+ concentration, external K+ concentration, and membrane potential. Rapid voltage-dependent transient pump currents are present in the mechanistic model, while they are absent in the ad hoc mathematical description we used. The stimulation results show that a sizable peak of pump current caused by the action potential upstroke in the mechanistic model affects phase 1 repolarization, and that this effect is relatively independent of the stimulation rate. The pump current generated by our ad hoc mathematical description is constant during the action potential and does not affect directly the repolarization time course. While the two Na,K pump models show similar pumping efficiency at low stimulation rates, the mechanistic pump is more efficient at high rates of activity. In essence, the distinctive features of the mechanistic model are due to an energy barrier expressing the voltage dependence of the translocation step of the mechanism, and to the redistribution of the intermediates of the biochemical reactions during activity. In comparison, the ad hoc mathematical description exhibits a fixed dependence of the pump current on voltage and ionic concentrations.

Identification of regulatory properties of metabolic networks by graph theoretical modeling.

An earlier graph theoretical model of metabolic and gene-expression networks has been modified and extended to include the effect of electrical potentials on binding constants, representation of uncatalyzed processes, and treatment of parallel reactions catalyzed by a single enzyme. Formal operations on the graph, which are facilitated by a set of standardized guidelines, identify the feedback signals in the network and rank them according to their influence. The technique was applied to a model of glycolysis in ascites tumor cells in the absence and presence of 12.5 mM exogenous glucose. Feedback regulation was widely distributed and mostly due to binding of adenine nucleotide cofactors to the enzymes of the network. The major changes in feedback regulation on adding glucose is the relief of inhibition of hexokinase and phosphofructokinase and the activation of pyruvate kinase. We conclude that regulation of tumor cell glycolysis is not restricted to hexokinase or to (Na+,K+)-ATPase as was previously suggested by others.

Simulation of the voltage dependence of the Na, K pump applied to cardiac cells.

We use simulation to study the dependence of the Na, K pump on membrane potential. Two consecutive mechanisms for the Na, K pump, based on a reduced Post-Albers scheme, are examined: one with six steps called GV3 and one with seven steps called MGV3. In GV3, a single voltage-dependent step combines both Na+ translocation and Na+ release into the extracellular medium. In MGV3, these two processes are allocated to two separate consecutive steps, but only the Na+ translocation step is voltage-dependent. Using the optimization software SCoPfit, numerical values of rate coefficients, symmetry factor (beta), and pump site density were found by fitting the models to published experimental data so that both GV3 and MGV3 could quantitatively reproduce steady-state current-voltage relationships for both forward and backward running of the pump, as well as [Na+]in and [K+]out activation curves. Using the rate coefficient values found by SCoPfit, we simulated a voltage-clamp experiment with both models running under their Na(+)-Na+ exchange mode, and we computed the transient currents generated following voltage steps in both depolarizing and hyperpolarizing directions from a basic potential of -40 mV. The voltage dependence of the rate constant (1/tau) of decay of the transient currents could qualitatively be reproduced when beta = 0.884 for GV3, and 0.932 for MGV3. The quantitative discrepancy between published experimental data and the theoretical curve generated by GV3 at potentials more negative than -20 mV was considerably reduced by using model MGV3. This finding alone suggests that a more detailed mechanism containing a single voltage-dependent step may reproduce all major steady-state and transient characteristics of the Na, K pump without the need of a second voltage sensitive step. However, the quantitative discrepancy between published experimental data and the theoretical curve generated by MGV3 at potentials more negative than -60 mV may be fully removed if either beta itself is voltage-dependent, or if a second voltage-dependent step is included in the model.

A model study of the contribution of active Na-K transport to membrane repolarization in cardiac cells.

A biochemical model of active Na-K transport in cardiac cells was studied in conjunction with a representation of the passive membrane currents and ion concentration changes. The active transport model is based on the thermodynamic and kinetic properties of a six-step reaction scheme for the Na,K-ATPase. It has a fixed Na:K stoechiometry of 3:2, and its activation is governed by three parameters: membrane potential intracellular Na+ concentration, and interstitial K+ concentration. The Na-K pump current is directly proportional to the density of Na,K-ATPase molecules. The passive membrane currents and ion concentration changes involve only Na+ and K+ ions, and no attempt was made to provide a precise representation of Ca2+ currents or Ca2+ concentration changes. The surface-to-volume ratio of the interstitial compartment is 55 times larger than that of the intracellular compartment. The flux balance conditions are such that the original equilibrium concentration values are re-established at each stimulation cycle. The underlying assumptions of the model were checked against experimental measurements on Na-K pump activity in a variety of preparations. In addition, the qualitative validation of the model was carried out by comparing its behavior following sudden frequency shifts to corresponding experimental observations. The overall behavior of the model is quite satisfactory and it is used to provide the following indications: (1) when the intracellular and interstitial volumes are relatively large, the ion concentration transients are small and the pumping rate depends essentially on average concentration levels. (2) An increase in internal Na+ concentration potentiates the response of the Na-K pump to rapid membrane depolarizations. (3) When the internal Na+ concentration is large enough, the Na-K pump current transient plays an important role in shaping the plateau and repolarization phase of the action potential. (4) A rapid increase in external K+ concentration during voltage clamp in multicellular preparations could saturate the Na-K pump response and lead to a fairly linear dependence of the pump activity on the internal Na+ concentration.

Childhood death and poverty: a study of all childhood deaths in Maine, 1976 to 1980.

All child deaths occurring from 1976 to 1980 in Maine were studied. All children who were participating in social welfare programs (Medicaid, Food Stamps, or Aid to Families with Dependent Children [AFDC]) at the time of death were categorized as children from "low-income" families. This group of children had an overall death rate 3.1 times greater than children who were not on a social welfare program at the time of death. Children from low-income families were at higher risk for disease-related deaths (3.5:1), accidental deaths (2.6:1), and homicide deaths (5.0:1), but not for suicides. These data suggest that excess mortality is occurring among infants and children from low-income families in spite of Medicaid and other poverty programs and that this excess mortality has important public health and social policy implications. Pediatricians and others interested in the well-being of children should support improvement of current health care delivery and social welfare programs, because the current system is failing to provide an optimal health outlook for every child.

Evaluation of a Behring laser-nephelometer prototype in the measurement of IgG, IgA and IgM.

A helium-neon laser nephelometer prototype manufactured by Hoechst Behring Institut was evaluated and used in our laboratory during a two-month period for the measurement of IgG, IgA and IgM. Two methods of dilution were usedto prepare standard curves. The best results were obtained when the patient serum was diluted one hundred fold (10-100 microliter) and incubated for 15 min at room temperature in a disposable plastic cuvette with the specific antiserum diluted five fold (100-300 microliter). The intensity of the light scattered by the antigen-antibody complexes was then immediately read on the digital voltmeter. The working ranges were 186-5944 mg/dl for IgG, 33-1066 mg/dl for IgA and 17-545 mg/dl for IgM. Standard curves obtained for the three immunoglobulins on six different days could be superimposed perfectly demonstrating an excellen reproducibility. The coefficient of variation for IgG (n = 8) was 4.1% at 5800 mg/dl. The results (n = 37) produced by the laser nephelometer (y) correlated very well with those obtained by radial immunodiffusion (x) for: IgG y = 8.07 + 1.00 X, r = 0.997, IgA y = - 6.51 + 1.05 X, r = 0.996, IgM y = 3.35 + 0.98 X, r = 0.998.