A solution to the problems of cytolysis assays with additional applications to other immunological and biochemical assays

After cellular immunoassays are compared with classical bioassays, conventional methods and consequent problems of data analysis for cytolysis assays are reviewed and a new solution is proposed. This solution incorporates new methods, called dose-response surface assays and analysis (DRSA), which estimate cytolytic activity coefficients on a surface in a three-dimensional space with two dose variables (killers and targets) and one response variable (counts). These new methods based on dose-response surfaces are demonstrated to be more informative and reliable than classical methods based on dose-response curves. In a test of the methods' robustness (sensitivity of parameter estimates to changes in the dose levels of the assay design), cytolytic activity coefficients estimated by DRSA varied by less than or equal to 30% over a reduction of three to four orders of magnitude in the dose levels. This remarkable robustness should be compared with the corresponding figures of as much as 500% over less than 1 order of magnitude for previously published results of coefficients estimated by conventional methods. DRSA is distinguished from replot-of-plots methods such as those used for enzyme inhibition assays in biochemistry, and is recommended as a more efficient method that should replace replot-of-plot methods now antiquated by the advent of microcomputers. DRSA can be applied to any experimental system that requires an activity coefficient to be estimated on a dose-response surface in a space of greater than or equal to 3 dimensions (greater than or equal to 2 dose variables and one response variable), regardless of the mathematical model and statistical estimators used to analyze the dose-response interaction. Finally, DRSA is compared with the methods known as response surface methodology (RSM), and is described as a new class of methods to be added to those that constitute RSM.     

On Blair's theory of excitation and the role of internal energy sources

It is shown that Blair's theory of excitation is independent of, and consequently valid for, any possible relationship between the threshold and the magnitude of the stimulus. It is pointed out that if a dependence of threshold on stimulus is assumed, the concept of rheobase becomes meaningless. Consequently contrary to Blair's impression, the disagreement between his theory in its original form and the experimental data on the time of incipient excitation with constant stimulus (response time) cannot be explained by assuming a dependence of the threshold on the magnitude of the stimulus. It is shown that a modification of Blair's interpretation, obtained by taking into account effects of internal energy sources released by the stimulus, eliminates the disagreement mentioned above between theory and experiment. The role of such modification in connection with propagation of excitation is discussed.     

Theoretical analysis of insulin-dependent glucose uptake heterogeneity in 3D bioreactor cell culture

Three-dimensional (3D) cell cultures in bioreactors are becoming relevant as models for biological and physiological in vitro studies. In such systems, mathematical models can assist the experiment design that links the macroscopic properties to single-cell responses. We investigated the relationship between biochemical stimuli and cell response within a 3D cell culture in scaffold with heterogeneous porosity. Specifically, we studied the effect of insulin on the local glucose metabolism as a function of 3D pore size distribution. The multiscale mathematical model combines the mass transport within a 3D scaffold and a signaling pathways model. It considers the scaffold heterogeneity, and it describes spatiotemporal concentration of metabolites, biochemical stimuli, and cell density. The signaling model was integrated into this model, linking the local insulin concentration at cell membrane to the glucose uptake rate through glucose transporter type 4 (GLUT4) translocation from the cytosol to the cell membrane. The integrated model determines the cell response heterogeneities in a single channel, hence the biological response distribution in a 3D system. It also provides macroscopic outcomes to evaluate the feasibility of an experimental measurement of the system response. From our analysis, it became apparent that the flow rate is the most important operative variable, and that an optimum value ensures a fast and detectable cell response. This model on insulin-dependent glucose consumption rate offers insight into the cell metabolism physiology, which is a fundamental requirement for the study metabolic disorder such as Type 2 diabetes mellitus, in which the physiological insulin-dependent glucose metabolism is impaired.     

Distortion of effects caused by indirect confounding

Undetected confounding may severely distort the effect of anexplanatory variable on a response variable, as defined by astepwise data-generating process. The best known type of distortion,which we call direct confounding, arises from an unobservedexplanatory variable common to a response and its main explanatoryvariable of interest. It is relevant mainly for observationalstudies, since it is avoided by successful randomization. Bycontrast, indirect confounding, which we identify in this paper,is an issue also for intervention studies. For general stepwise-generatingprocesses, we provide matrix and graphical criteria to decidewhich types of distortion may be present, when they are absentand how they are avoided. We then turn to linear systems withoutother types of distortion, but with indirect confounding. Forsuch systems, the magnitude of distortion in a least-squaresregression coefficient is derived and shown to be estimable,so that it becomes possible to recover the effect of the generatingprocess from the distorted coefficient.     

Mechanism of metabolic adaptation

The paper presents results of investigations on some mechanisms of metabolic adaptation in mammals. There are four sections in the tissue metabolic system of acid-base homeostasis: polyamines as factors of metabolic adaptation; significance of carbon dioxide for metabolic response formation in hypobiosis; polyamines metabolism in hypobiosis. Peculiarities of intermediate metabolism have been analyzed in animal tissues under the changes in H+, CO2 and HCO3- concentrations. Basing on a new interpretation of the experimental data and detected regularity in the metabolism, conclusion on the existence of a new acid-base homeostasis system in the tissues has been made. The results of polyamines metabolism investigations in the mammals under the stress have been described. The experimental data make us to believe that changes in polyamines synthesis and ODC activity in particular, is a part of stereotype nonspecific response to any stress impacts and one of the factors of cell metabolic adaptation. Some new data on mechanisms of formation and control of metabolic status of animals in the natural and artificial hibernation have been presented. The key idea is that in the state of hypobiosis the carbon dioxide (HCO3-) appears as a regulatory factor of metabolic adaptation, which is able to realize its action directly via affecting numerous biochemical events. The participation of polyamines in adaptive metabolic response to hybernation factors is suggested. Some peculiarities of ornithine decarboxilase and transglutaminase activity during the different stages of genuine and artificial hypobiosis have been demonstrated.     

Nonlinear optical studies of heme protein dynamics: implications for proteins as hybrid states of matter

Protein structure is fundamentally related to function. However, static structures alone are insufficient to understand how a protein works. Dynamics play an equally important role. Given that proteins are highly associated aperiodic systems, it may be expected that protein dynamics would follow glass-like dynamics. However, protein functions occur on time scales orders of magnitude faster than the time scales typically associated with glassy systems. It is becoming clear that the reaction forces driving functions do not sample entirely the large number of configurations available to a protein but are highly directed along an optimized pathway. Could there be any correlation between specific topological features in protein structures and dynamics that leads to strongly correlated atomic displacements in the dynamical response to a perturbation? This review will try to provide an answer by focusing upon recent nonlinear optical studies with the aim of directly observing functionally important protein motions over the entire dynamic range of the protein response function. The specific system chosen is photoinduced dynamics of ligand dissociation at the active site in heme proteins, with myoglobin serving as the simplest model system. The energetics and nuclear motions from the very earliest events involved in bond breaking on the femtosecond time scale all the way out to ligand escape and bimolecular rebinding on the microsecond and millisecond time scale have been mapped out. The picture that is emerging is that the system consists of strongly coupled motions from the very instant the bond breaks at the active site that cascade into low frequency collective modes specific to the protein structure. It is this coupling that imparts the ability of a protein to function on time scales more commensurate with liquids while simultaneously conserving structural integrity akin to solids.     

Cyclic 3'', 5''-AMP relay in dictyostelium discoideum. IV. Recovery of the cAMP signaling response after adaptation to cAMP

In dictyoselium discoideum, an increase in extracellular cAMP activates adenylate cyclase, leading to an increase in intracellular cAMP and the rate of cAMP secretion. Cells adapt to any constant cAMP stimulus after several minutes, but still respond to an increase in the concentration of the stimulus. We have now characterized the decay of adaptation (deadaptation) after the removal of cAMP stimuli. Levels of adaptation were established by the perfusion of [(3)H]adenosine-labeled amoebae with a defined cAMP stimulus. After a variable recovery period, the magnitude of the signaling response to a second stimulus was measured; its attenuation was taken as a measure of residual adaption to the first stimulus. The level of adaptation established by the first stimulus depended on both its magnitude and duration. Deadaptation began as soon as the first stimulus was removed. The magnitude of the response to the second stimulus increased with the recovery time in a first-order fashion, with a t(1/2)=3-4 min for stimuli of 10(-8) M to 10(-5) M cAMP. Responses to test stimuli, although reduced in magnitude, had an accelerated time-course when they closely followed a prior response that had not completely subsided. This effect is called priming; we believe it reveals a reversible, rate-limiting step that modulates the onset and termination of the signaling responses of amoebae that have not recently responded to a cAMP stimulus. We have suggested that the cAMP signaling response is controlled by two antagonistic cellular processes, excitation and adaptation. The data reported here imply that both the rate of rise in the adaptation process and the final level reached depend on the occupancy of cAMP surface receptors and that the decay of adaptation when external cAMP is removed proceeds with first-order kinetics.     

Pharmacogenetics: Implications for drug development,patients and society

Clinical diagnosis and prescribing is a highly sophisticated art, requiring many years of training. Despite this, the response of individual patients to medicines (where efficacy and safety have been proven in large clinical studies) can still be somewhat variable. Knowledge of the likely response of an individual patient to a medicine will enable physicians to select the most effective and well-tolerated treatment for that patient. Pharmacogenetics is the use of genetic science and technology to provide new insights on the likely response to a particular medicine. (This contrasts with the more conventional use of genetics to elicit information about diseases.) Pharmacogenetic medicine response profiles could take the form of either gene-specific profiles, which will determine the gene variants that affect the mode of action and the metabolism of the medicine, or abbreviated single nucleotide polymorphism profiles, which are correlated with medicine-related phenotypes. In general, pharmacogenetic medicine response profiles will be unlikely to provide additional information about the patient's disease or predict any other diseases. The ethical, legal and social issues associated with medicine response profiles are clearly of a quite different magnitude from those associated with the gene-specific tests for disease.     

Mapping functional similarity of predators on the basis of trait similarities

Theoretical and empirical studies in community ecology often simplify their study system by lumping together species on the basis of trait similarities (e.g., their taxonomy, resource or microhabitat usage) and then assume species sharing similar traits are functionally similar. To date, no study has directly tested whether species more similar with respect to any of these traits are more similar in their functional effects on population or ecosystem processes. In this study, we examined the association between traits and functional effects of six different aquatic predatory vertebrates. We demonstrated that functional similarity across multiple response variables was not correlated with trait similarity, but differences in trait values were associated with significantly different effects on individual response variables. The exact relationship between species traits and functional effect of predators, however, was different for each response variable. Using traits to predict functional similarity among species may be useful when considering individual response variables, but only if it is known which traits have the greatest influence on the response variable of interest. It is doubtful that any one scheme will predict the functional similarity of species across a diverse array of response variables because each response will likely be strongly influenced by different traits.     

The regulation of phosphoenolpyruvate carboxylase in CAM plants

Phosphoenolpyruvate carboxylase catalyses the primary assimilation of CO(2) in Crassulacean acid metabolism plants. It is activated by phosphorylation, and this plays a major role in setting the day-night pattern of metabolism in these plants. The key factor that controls the phosphorylation state of phosphoenolpyruvate carboxylase is the activity of phosphoenolpyruvate carboxylase kinase. Recent work on Crassulacean acid metabolism plants has established this enzyme as a novel protein kinase and has provided new insights into the regulation of protein phosphorylation. Phosphoenolpyruvate carboxylase kinase is controlled by synthesis and degradation in response to a circadian oscillator. The circadian control of phosphoenolpyruvate carboxylase kinase can be overridden by changes in metabolite levels. The primary effect of the circadian oscillator in this system may be at the level of the tonoplast, and changes in kinase expression may be secondary to circadian changes in the concentration of a metabolite, perhaps cytosolic malate.     

Adenylate cyclase and phosphodiesterase activities in rat hepatocytes cultured in the presence and absence of dexamethasone

The effects of glucagon and dexamethasone on the activities of the enzymes involved in cyclic adenosine 3':5'-monophosphate (cyclic AMP) metabolism in primary monolayer cell cultures of adult rat hepatocytes were examined. Short-term experiments indicated that the magnitude of the cultured cells' response to glucagon, as measured by production of cyclic AMP, was essentially the same as that for freshly isolated hepatocytes. However, the time course of this response was markedly different. Although the activity of adenylate cyclase is maintained throughout the culture period at a level similar to that of the freshly isolated hepatocytes, the activity of both low and high Km forms of phosphodiesterase decreases rapidly with length of time in vitro. This is reflected by an increase in cyclic AMP produced in response to glucagon and theophylline by cells of different ages. Dexamethasone caused an increased loss of phosphodiesterase activity, as well as increased cyclic AMP accumulation in the presence or absence of theophylline. Various agents failed to restore the lost phosphodiesterase activity. These results may indicate that phosphodiesterase activity is more sensitive to the inevitable inadequacies of the in vitro environment of cultured hepatocytes than adenylate cyclase. It was also found that a modification of the method of Seglen (1) for the preparation of isolated hepatocytes yielded cells that had less phosphodiesterase activity than those prepared by the method of Berry and Friend (2).     

The influence of the cost of growth on ectotherm metabolism

A new model of ectothermic growth and metabolism is proposed. This model differs from most earlier models in representing explicitly the contribution of the "cost of growth" to ectotherm metabolism. It is shown that the cost of growth may constitute between 17 and 29% of the metabolism of an "average" ectotherm population. Furthermore, the metabolism of an "average" growing ectotherm may be between 40 and 79% greater than that of a non-growing ectotherm. As many environmental factors induce changes in metabolic rates of this magnitude, it is suggested that many factors which cause changes in metabolic rates do so indirectly by altering growth rates. In particular, it is suggested that body size, food availability and temperature often indirectly influence metabolic rates through their effects on growth rates, rather than by directly determining metabolic rates as has usually been assumed.     

On the dispersion index of a Markovian molecular clock

The number of nucleotide substitutions accumulated in a gene or in a lineage is an important random variable in the study of molecular evolution. Of particular interest is the ratio of the variance to the mean of that random variable, often known as the dispersion index. Because nucleotide substitution is most commonly modeled by a continuous-time four-state Markov chain, this paper provides a systematic method of computing the dispersion indices exhibited by a continuous-time four-state Markov chain. Using this method along with computer algebra and Monte Carlo simulation, this paper offers partially proven conjectures that were supported by thorough computer experiments. It is believed that the Tamura model, the equal-input model and the Takahata-Kimura model always exhibit dispersion indices less than 2. It is also believed that a general four-state model can be chosen to exhibit a dispersion index of any desired magnitude, although the chance of a randomly chosen such model exhibiting a dispersion index greater than 2 is as small as about 2%. Relevance of these findings to the neutral theory is discussed.     

Parameter identification of the human lower limb under dynamic, transient torsional loading

The response of the lower limb to dynamic, transient torsional loading applied at the foot has been measured for a male test subject. The dynamic loading was provided by a computer controlled pneumatic system which applied single haversine (i.e. half cycle of a sine wave) axial moment pulses of variable amplitude (0-100 Nm) and duration (50-600 ms). Potentiometers measured the absolute rotations of the three leg segments. Test variables included rotation direction, weight bearing and joint flexion. Two approaches were explored for specifying parameters (i.e. inertia, damping, stiffness) of a three degree-of-freedom dynamic system model which best duplicated the measured response. One approach involved identification of linear parameters by means of optimization while the other approach entailed estimation. Parameter estimates, which included non-linear, asymmetric stiffness functions, were derived from the literature. The optimization was undertaken so as to identify parameter dependence on test variables. Results indicate that parameter values are influenced by test variables. Results also indicate that the non-linear, estimated model better approximates the experimental data than the linear, identified model. In addition to identifying parameters of a three degree-of-freedom model, parameters were also identified for a single degree-of-freedom model where the motion variable was intended to indicate the rotation of the in vivo knee. It is concluded that the simpler model offers good accuracy in predicting both magnitude and time of occurrence of peak knee axial rotations. Model motion fails to track the measured knee rotation subsequent to the peak, however.     

The effect of fasting on activity and resting metabolism in the sand cricket, Gryllus firmus: a multivariate approach

Heat increment of feeding (HIF) is a ubiquitous feature of animals, and corresponds to a conspicuous rise in metabolism after a meal, induced by the release of energy due to digestion and absorption of foodstuffs. However, there exists great variation both in the duration and magnitude of HIF. In insects, HIF is well known, and it appears to be dramatic, especially in immature stages. However, little is known about the effect of HIF on different aspects of metabolism. We determined metabolic rate as CO2 production in fasted and non-fasted nymphs of the sand cricket (Gryllus firmus). A number of metabolic variables were computed from the simultaneous activity record: activity, resting, minimum, maximum and average metabolic rate. Our results suggest that there is a general effect of fasting in metabolic rate but with a graded response: the larger the influence of activity on the metabolic variable, the less is the effect of fasting that was detected.     

An efficient method for calculation of dynamic logarithmic gains in biochemical systems theory

Biochemical systems theory (BST) characterizes a given biochemical system based on the logarithmic gains, rate-constant sensitivities and kinetic-order sensitivities defined at a steady state. This paper describes an efficient method for calculation of the time courses of logarithmic gains, i.e. dynamic logarithmic gains L(Xi, Xj; t), which expresses the percentage change in the value of a dependent variable Xi at a time t in response to an infinitesimal percentage change in the value of an independent variable Xj at t=0. In this method, one first recasts the ordinary differential equations for the dependent variables into an exact canonical nonlinear representation (GMA system) through appropriate transformations of variables. Owing to the structured mathematical form of this representation, the recast system can be fully described by a set of numeric parameters, and the differential equations for the dynamic logarithmic gains can be set up automatically without resource to computer algebra. A simple general-purpose computer program can thus be written that requires only the relevant numeric parameters as input to calculate the time courses of the variables and of the dynamic logarithmic gains for both concentrations and fluxes. Unlike other methods, the proposed method does not require to derive any expression for the partial differentiation of flux expressions with respect to each independent variable. The proposed method has been applied to two kinds of reaction models to elucidate its usefulness.     

Phenomenological definition of response times with application to metabolic reactions

The metabolic response time, i.e. the delay the system introduces in the response to an input flux, is considered. A novel phenomenological definition is presented, which is valid for any kind of behavior, including transitory or permanent oscillatory responses. In order to calculate the response time of single-input systems, output fluxes have to be deconvoluted with the input flux. The bases for this are established. The resulting function (unit impulse response in time-invariant linear systems) is transformed by subtracting its final state, taking the absolute value and normalizing by the resulting area, so that a norm can be applied that weights the response at every time. This response time can also be interpreted as an average. It coincides with the transition (characteristic) time of an output flux, provided that the input is performed instantaneously (step function). A strictly non-negative response function is needed for the response time to be interpreted as a mass balance. A simple example is used to study the deviation otherwise. The method is advantageous in that it provides clues on the phenomenological behavior of biochemical systems. For example, deconvolution reveals the intrinsic oscillation-generating mechanism of an allosteric enzyme, which becomes hidden when the input flux increases in a slow way. This is illustrated by means of a model.     

Cholesterol sulfate is a naturally occurring inhibitor of steroidogenesis in isolated rat adrenal mitochondria

We previously reported (Lambeth, J. D., Xu, X. X., and Glover, M. (1987) J. Biol. Chem. 262, 9181-9188) that exogenously added cholesterol sulfate inhibits the conversion of cholesterol to pregnenolone in isolated adrenal mitochondria, and does so by affecting intramitochondrial cholesterol movement but not its subsequent metabolism to pregnenolone by cytochrome P-450scc. We now report that a major kinetic component of the inhibition is noncompetitive with respect to cholesterol, consistent with an allosteric effect at a site other than the substrate binding site of cytochrome P-450scc. We now also report that cholesterol sulfate is present as an endogenous compound in preparations of adrenal mitochondria. Its content varied from 0.05 to 0.8 nmol/mg protein. Cholesterol sulfate level correlated inversely with the mitochondrial cholesterol side-chain cleavage activity. Endogenous cholesterol sulfate thus appeared to account for the variable rates of pregnenolone synthesis which were seen in different mitochondrial preparations. Cholesterol sulfate was metabolized to pregnenolone sulfate by a mitochondrial side-chain cleavage system, but proved to be a relatively poor substrate for an extramitochondrial steroid sulfatase activity present in adrenal cortex. Confirming a role as a naturally occurring inhibitor, removal of endogenous mitochondrial cholesterol sulfate by metabolism to pregnenolone sulfate correlated with a 3-fold activation of cholesterol side-chain cleavage. We suggest that cholesterol sulfate functions in steroidogenic tissues to regulate the magnitude of the steroidogenic response.     

Complementary idiotypy in the regulation of the immune response.

A new concept is presented for the interactions of two complementary antibodies in the immune response. These antibodies bind to each other by means of their variable sequence determinants and therefore are designated as complementary idiotypes. Under certain conditions, both complementary idiotypes are produced by the same animal at the same time. An idiotype can drastically affect the expression of the complementary idiotype in the animal, inducing a peripheral quench effect of antibody-binding activity and a central effect on the immunocompetent cell, which produces the complementary idiotype. It is proposed that complementary idiotypes might be induced during every immune response, thus playing an essential role in the regulation of the immune response.