Application of the avidin-biotin system for post-embedding cytochemical demonstration of a biotin-labeled IgG tracer

The capacity of the avidin-biotin system for post-embedding cytochemical detection of a biotin-labeled proteinaceous tracer was investigated. By testing modifications of the fixatives, the epoxy embedding medium, and various etching solutions, a procedure was developed to localize specifically the biotinylated tracer on 1-micron thick and thin sections. By use of this technique, a systemically administered IgG tracer was demonstrated after 24 hr throughout the endomysium of mouse skeletal muscle. In the adjoining sciatic nerve, the tracer IgG occurred at the perineurium and within endoneurial blood vessels, although the endoneurium itself was spared because of the presence of the blood-nerve barrier. Because of the small size of the biotin ligand and the non-denaturing method of labeling proteins, our mode of application of the avidin-biotin system appears suitable for tracer studies.     

Violation of the fluctuation-dissipation theorem in a protein system

We report the results of molecular dynamics simulations of the protein myosin carried out with an elastic network model. Quenching the system, we observe glassy behavior of a density correlation function and a density response function that are often investigated in structure glasses and spin glasses. In the equilibrium, the fluctuation-response relation, a representative relation of the fluctuation-dissipation theorem, holds that the ratio of the density correlation function to the density response function is equal to the temperature of the environment. We show that, in the quenched system that we study, this relation can be violated. In the case that this relation does not hold, this ratio can be regarded as an effective temperature. We find that this effective temperature of myosin is higher than the temperature of the environment. We discuss the relation between this effective temperature and energy transduction that occurs after ATP hydrolysis in the myosin molecule.     

Evidence for a generalized Laguerre transform of temporal events by the visual system

It is generally assumed that the early visual processing is constituted by a set of filters operating in parallel. In this respect the visual system performs a transform, generating a code of the characteristics of the input signal. Recently, it has been suggested that the coding of the spatial characteristics by the visual system can be described by a Hermite transform (Martens, 1990a, b). It was also suggested that a three-dimensional Hermite transform can be used to code spatiotemporal events. In contrast to this latter suggestion, we argue that the coding of temporal events takes the form of a generalized Laguerre transform. We review psychophysical evidence supporting this hypothesis.     

A generalized model of a resource-population system

Summary A system of nonlinear differential equations describing a resource-population system is analyzed in terms of the existence and characteristics of its equilibrium states.It is proved that, under the condition that k< (necessary condition for the population being able to grow under optimal conditions), it is a necessary and sufficient condition for the system to have a steady state that the resource input rate to the system be constant.When the resource input rate is a constant different from zero, the system has only one equilibrium point, at M ⁰=/⁰/k, A ⁰=–(/⁰/k)ln(1–k/), and this equilibrium point is always stable. In other words, the system population-resource will always reach the steady state, either monotonically (node) or by damped oscillations (focus), from any arbitrary initial condition in the positive quadrant.When the resource input rate is equal to zero, the system has an infinite number of equilibrium points at M ₀=0, A ₀=constant. All these equilibrium points are unstable in the sense that any slight increase in M will move the system away from the equilibrium states, except for the point M ₀=0, A ₀=0, which is the only stable equilibrium point, to which the system will tend. This stable equilibrium point corresponds to the condition of complete annihilation of both resource and population.Finally, it is proved that the system does not have limit cycles in the positive quadrant and is therefore incapable of self-oscillations.This work was partially supported by a Ford Foundation fellowship and various Cornell University fellowships.     

Normalization in the fitting of data by iterative methods. Application to tracer kinetics and enzyme kinetics

1. The normalization of biochemical data to weight them appropriately for parameter estimation is considered, with reference particularly to data from tracer kinetics and enzyme kinetics. If the data are in replicate, it is recommended that the sum of squared deviations for each experimental variable at each time or concentration point is divided by the local variance at that point. 2. If there is only one observation for each variable at each sampling point, normalization may still be required if the observations cover more than one order of magnitude, but there is no absolute criterion for judging the effect of the weighting that is produced. The goodness of fit that is produced by minimizing the weighted sum of squares of deviations must be judged subjectively. It is suggested that the goodness of fit may be regarded as satisfactory if the data points are distributed uniformly on either side of the fitted curve. A chi-square test may be used to decide whether the distribution is abnormal. The proportion of the residual variance associated with points on one or other side of the fitted curve may also be taken into account, because this gives an indication of the sensitivity of the residual variance to movement of the curve away from particular data points. These criteria for judging the effect of weighting are only valid if the model equation may reasonably be expected to apply to all the data points. 3. On this basis, normalizing by dividing the deviation for each data point by the experimental observation or by the equivalent value calculated by the model equation may both be shown to produce a consistent bias for numerically small observations, the former biasing the curve towards the smallest observations, the latter tending to produce a curve that is above the numerically smaller data points. It was found that dividing each deviation by the mean of observed and calculated variable appropriate to it produces a weighting that is fairly free from bias as judged by the criteria mentioned above. This normalization factor was tested on published data from both tracer kinetics and enzyme kinetics.     

The differential equations for specific activities in a distributed tracer system

Under the assumption of simple Fick-law diffusion with convection and/or imposed force fields, the flux law for tagged molecules in a tracer system is derived, and the Sheppard-Householder interfusion coefficient identified. The partial differential equations for concentrations of tagged species and for specific activities in an open distributed reaction system are derived and compared.     

A tracer method to study unidirectional fluxes of lithium: Application to frog skin

We describe a new tracer method to measure unidirectional fluxes of Li⁺, despite the lack of any utilizable radioisotope of lithium. This method uses the purified stable isotopes, ⁶Li and ⁷Li, detected with an ion-probe microanalyser. The accuracy is comparable to that obtained for other ions (e.g., Na⁺) with radiotracers.The method has been applied to frog skin with both faces bathed in a 20% lithium/80% sodium medium. Sodium and lithium unidirectional fluxes have been measured simultaneously. The results are consistent with lithium being actively pumped, the outflux of lithium being, however, much larger than that of sodium.     

Basic studies on metabolic steady state. Linearity of a metabolic tracer system

Summary In dogs the changes in the plasma concentration following the injection and/or infusion of labeled glucose reveal the labeled glucose system to be a linear system. Based on a simple chemical model it was shown that the rate of removal from the system of any tracer injected as single impulse can be described by a first order chemical reaction, even in systems from which the tracee is removed by a process of a higher order, provided the tracee is in the steady state and the concentration of tracer in the plasma is low compared to that of the tracee. The validity of the commonly used formulae for the calculations of the rates of disappearance from systems in a steady state is based on this first order process.     

Application of a biological tracer in transporting water volumes during eutrophication of mondsee

The oligotrophic alpine lake Mondsee belonging to the system of eight lakes in the Salzkammergut region in Austria represented during our investigation the plankton distribution model in those parts of water areas that are situated in front of the estuaries of intensely eutrophicated mountain inflow torrents, such as Wangauer Ache and Fuschler Ache. Simultaneously with water sampling, measurements by drift floats were made to detect the water inflow direction and speed at several depths down to the bottom. The results have shown that horizontal as well as vertical plankton movement is an efficient biotracer for water flow parameters. Samples were collected in a “chessboard” way and also vertically at 5 m distances. Zoo-, phyto- and bacterioplankton were evaluated.     

Isolation of bright aggregate fluctuations in a multipopulation image correlation spectroscopy system using intensity subtraction

Image correlation spectroscopy allows sensitive measurement of the spatial distribution and aggregation state of fluorescent membrane macro molecules. When studying a single population system (i.e., aggregates of similar brightness), an accurate measure can be made of the aggregate number per observation area, but this measurement becomes much more complex in a distributed population system (i.e., bright and faint aggregates). This article describes an alternate solution that involves extraction of the bright aggregate population information. This novel development for image correlation spectroscopy, termed intensity subtraction analysis, uses sequential uniform intensity subtraction from raw confocal images. Sequential intensity subtraction results in loss of faint aggregate fluctuations that are smaller in magnitude than fluctuations due to the brightest aggregates. The resulting image has correlatable fluctuations originating from only the brightest population, permitting quantification of this population's distribution and further cross-correlation measurements. The feasibility of this technique is demonstrated using fluorescent microsphere images and biological samples. The technique is further used to examine the spatial distribution of a plasma-membrane-labeled fluorescent synthetic ganglioside, and to cross-correlate this probe with various membrane markers. The evidence provided demonstrates that bright aggregates of the fluorescent ganglioside are associated with clathrin-coated pits, membrane microvilli, and detergent-resistant membranes.     

Exploitation of a deep-water algal maximum by Daphnia: a stable-isotope tracer study

The exploitation of a deep algal maximum by Daphnia in the absence of fish predation was studied in large indoor mesocosms. Facing the dilemma of low food but high temperature in the epilimnion vs. high food but low temperature in the hypolimnion, Daphnia distribute above and below the thermocline in order to optimise their fitness. Labelling hypolimnetic algae with ¹⁵N revealed that the vertical distribution of Daphnia is dynamic, i.e., all individuals traverse the thermocline and allocate a certain proportion of their time to feeding in the cold water. The overall energy gain from the deep-water algal maximum is lower than from the same algal concentration in the epilimnion due to the low temperature and the limited time an individual spends in the hypolimnion. The results provide mechanistic support for the hypothesis that Daphnia chose their habitat according to an Ideal Free Distribution with Costs model.     

Application of a taste evaluation system to the monitoring of Kimchi fermentation

As an objective method, taste evaluation with an instrument is able to supplement the subjective sensory evaluation and to be applied to the optimization of food processing. Kimchi, a Korean traditional pickle fermented with lactic acid bacteria, is expanding its consumption worldwide. The fermentation control of it has been routinely done by measuring titratable acidity and pH. In this study, an eight-channel taste evaluation system was prepared, followed by an application to the monitoring of Kimchi fermentation. Eight polymer membranes which individually responded to cationic or anionic substances were prepared by mixing electroactive materials such as tri-n-octylmethylammonium chloride, bis(2-ethylhexyl)sebacate as the plasticizer and polyvinyl chloride in the ratio of 1:66:33. Each membrane prepared was separately installed onto the sensitive area of an ion-selective electrode to produce the respective taste sensor. The eight-channel sensor array and a double junction reference electrode were connected to a 16-channel high input impedance amplifier. The amplified sensor signals were stored to a personal computer via a multi-channel A/D converter. Two sensor groups composed of the cation-selective and anion-selective polymer membrane electrodes showed characteristic concentration-dependency to various artificial taste substances. As a whole, the response potentials of the sensor array increased during the fermentation period at 4, 10 and 25 degrees C. Even the response potentials of the anion-selective taste sensors slightly increased possibly due to the protonation of anions by liberated H+ ions, thereby leading to a decrease in the anion concentration. When the signal data were interpreted by principal component analysis (PCA), the first PC at 4 degrees C explained most of the total data variance. A close correlation was found between the values of titratable acidity and the first PC, which indicated a possible applicability of the multi-channel taste sensor of this study to the process monitoring of various pickle.     

Backwrd population projection by a generalized inverse

The Leslie population projection matrix may be used to project forward in time the age distribution or age-sex distribution of a population. As it is a singular matrix, it does not have an inverse, and so it is not clear that there is a corresponding procedure for backward projection. In terms of the eigenvalues and eigenvectors of the Leslie matrix, certain generalized inverses are constructed that can sometimes be used advantageously for backward projection.