Diffusion profiles of Na+-fluorescein in frog ventricular muscle.

Frog ventricular muscle strips were placed in a single sucrose-gap chamber to measure the interdiffusion of solutes across the sucrose-Ringer's solution partition. Steady-state diffusion profiles of fluorescein sodium developed along the axis of the muscle in the physiological node by continuously perfusing the sucrose pool with 210-mM sucrose plus fluorescein (5-10 mM). Fluorescein was found to diffuse freely through the extracellular space of the ventricular muscle without binding to the tissue. The fluorescence of Na+-fluorescein in the muscle (measured at 530 +/- 30 nm) varied linearly with the dye concentration in the sucrose perfusate. The diffusion profiles of dye in the test node depended on the tightness or snaring of the muscle strip by the latex diaphragms, the diameter of the muscle strip, and changes in hydrostatic pressure between the sucrose and Ringer's solution pools. Fluorescein concentration in the cross section of test node closest to the latex partition (sucrose-Ringer's solution interface) ranged between 4-13% of the dye concentration in the sucrose pool. These values are more than five times smaller than those estimated theoretically, assuming free diffusion. The experimental findings indicate that the presence of a physical barrier, such as a rubber diaphragm, limits free interdiffusion of solutes across the sucrose gap. The presence of such a barrier thus prevents large concentration gradients from occurring in the extracellular spaces along the physiological node.     

The correction factors for sucrose gap measurements and their practical applications.

The distribution of extracellular and intracellular potential in the sucrose gap apparatus, previously established for a single fiber using the cable equations for a core conductor model (Jirounek and Straub, Biophys. J., 11:1, 1971), is obtained for a multifiber preparation. The exact equation is derived relating the true membrane potential change to the measured potential differences across the sucrose gap, the junction potentials between sucrose and physiological solution, the membrane potential in the sucrose region, and the electrical parameters of the preparation in each region of the sucrose gap. The extracellular potential distribution has been measured using a modified sucrose gap apparatus for the frog sciatic nerve and the rabbit vagus nerve. The results indicate a hyperpolarization of the preparations in the sucrose region, of 60--75 mV. The hyperpolarization is independent of the presence of junction potentials. The calculation of the correction terms in the equation relating the actual to the measured potential change is illustrated for the case of complete depolarization by KC1 on one side of the sucrose gap. The correction terms in the equation are given for various experimental conditions, and a number of nomographic charts are presented, by means of which the correction factors can be rapidly evaluated.     

Reexamination of the double sucrose gap technique for the study of lobster giant axons. Theory and experiments

The double sucrose gap technique for the study of lobster giant axons has been reexamined. The leakage behavior of the system cannot be successfully modeled by conventional sucrose gap theory, but is accounted for by the McGuigan-Tsien model that takes into account the cable properties of membrane under sucrose. The facts of high-leakage conductance and the ability to maintain large resting potentials in the face of low sucrose gap resistance lead to a hypothesis that membrane resistance under sucrose is very low because of a large negative surface potential. Computer simulations of the leakage behavior of the conventional gap model and the McGuigan-Tsien model were compared with experimental measurements on lobster axons using normal sucrose or sucrose doped with Na+, Ca2+ or La3+ ions. As the concentration of doping ion increased, the leakage rose, but the species of doping ion had more influence on leakage than gap resistance. At equal gap resistance, leakage decreased with an increase in valence of the doping species. Leakage was even lower in La-doped sucrose at 20 M omega gap resistance than in normal sucrose at 200 M omega gap resistance. Resting potentials decreased with decreasing gap resistance and increasing valence of the doping species. Resting potential behavior was successfully simulated with a hybrid model consisting of a point node flanked by infinite cables and a shunt between ground and the voltage-measuring pool. The data support the hypothesis that the membrane resistance under sucrose is low and that it can be raised by doping the sucrose with multivalent cations, with La3+ being particularly effective.(ABSTRACT TRUNCATED AT 250 WORDS)     

A theoretical study on the sucrose gap technique as applied to multicellular muscle preparations. II. Methodical errors in the determination of outward currents.

This paper presents a mathematical model for analyzing systematic errors associated with the membrane conductance of multicellular muscle preparations as determined in a sucrose gap apparatus. The errors arise because of the interdiffusion of sucrose and saline in the interstitial fluid spaces, which results (a) in spatial variations of equilibrium potentials, membrane conductance, and solution conductivity, and (b) in the existence of a liquid junction potential. The model was applied to simulate the measurement of outward currents predominantly carried by potassium ions; time variations were not considered. Output current/voltage (I/V) curves were computed and compared with the membrane I/V relationship used in the computation. The output curves look very much like experimental results but are distorted considerably from the membrane I/V relationship: (a) under favorable conditions (negligible shunt current), the membrane current is overestimated over the entire range of membrane potential, (b) regions with negative slope conductance of I/V relations with "anomalous rectifier" properties are found to be less pronounced or even absent, and (c) resting potentials may be either increased or reduced. The origin of these errors is related to currents emerging from the sucrose compartment (local circuit as well as externally applied currents). Their dependence on several experimental parameters is discussed.     

Analysis of lumped and distributed elements models of cut muscle fibers in vaseline or sucrose gap preparations

A general method of finding the time course and the steady state distribution of potential in Vaseline or sucrose gap preparations is given by making use of the linear cable equation. The general solution has been found analytically in terms of its Laplace transform and then numerically inverted. Two particular experimental situations, namely the single gap and the double gap preparations, have been analyzed. The results have been compared with the solutions of the commonly used lumped elements models. While for the double gap no large errors are introduced by the lumped model, for the single gap there are significant differences. The dependence of the voltage distribution on various electrical and geometrical parameters has been examined. It is suggested that the proposed mathematical treatment might be used by experimenters as a reference to assess the validity of simplified lumped models.     

Permeation of ammonia across bilayer lipid membranes studied by ammonium ion selective microelectrodes.

Ammonium ion and proton concentration profiles near the surface of a planar bilayer lipid membrane (BLM) generated by an ammonium ion gradient across the BLM are studied by means of microelectrodes. If the concentration of the weak base is small compared with the buffer capacity of the medium, the experimental results are well described by the standard physiological model in which the transmembrane transport is assumed to be limited by diffusion across unstirred layers (USLs) adjacent to the membrane at basic pH values (pH > pKa) and by the permeation across the membrane itself at acidic pH values. In a poorly buffered medium, however, these predictions are not fulfilled. A pH gradient that develops within the USL must be taken into account under these conditions. From the concentration distribution of ammonium ions recorded at both sides of the BLM, the membrane permeability for ammonia is determined for BLMs of different lipid composition (48 x 10(-3) cm/s in the case of diphytanoyl phosphatidylcholine). A theoretical model of weak electrolyte transport that is based on the knowledge of reaction and diffusion rates is found to describe well the experimental profiles under any conditions. The microelectrode technique can be applied for the study of the membrane permeability of other weak acids or bases, even if no microsensor for the substance under study is available, because with the help of the theoretical model the membrane permeability values can be estimated from pH profiles alone. The accuracy of such measurements is limited, however, because small changes in the equilibrium constants, diffusion coefficients, or concentrations used for computations create a systematic error.     

Ribosomal dysentery vaccine. I. Method of production, physical and chemical properties]

Studies of A. and G. Youmans on the experimental tuberculosis led to discovery of a fundamentally new type of vaccines (ribosomal vaccines) which proved to be highly effective in the prophylaxis of many experimental infections. Therefore it seems reasonable to prepare analogous vaccine from Shigellae for the study of its efficiency in experimental shigellosis. Ribosomal preparations from Shigella sonnei were prepared by sonic disruption of microbial cells followed by differential ultracentrifugation according to A. and G. Youmans' method with slight modifications. The yeild of ribosomal fraction was about 2 per cent by weight; all the series had an UV adsorption maximum at 260 nm, the ratio OD260:OD280 being approximately 2. They contained about 55% of RNA, 35% of protein and no more than 8% of saccharides. As shown by centrifugation in sucrose gradient and by analytical ultracentrifugation the preparations were homogeneous. The presence of undissociated ribosomes was confirmed by electron microscopy. Thus, the ribosomal preparations obtained proved to be sufficiently purified for carrying out experimental investigations of their biological activity.     

Studies of Impedance in Cardiac Tissue Using Sucrose Gap and Computer Techniques: I. The Influence of Sucrose and Oil as Insulating Media

Impedances of cardiac cells of an insect were determined as a function of time to test the effects of sucrose and oil as insulating media in a gap arrangement. Impedance values are shown to increase markedly with time when sucrose is used as the insulating agent. Although impedance values are steady when oil is used, it is suggested that a layer of trapped electrolyte provides a shunt pathway and seriously impairs the validity of the measurements. A quick wash with sucrose followed by oil does not alleviate the situation but leaves a layer of sucrose trapped at the tissue-medium interface into which ions diffuse. The hypotheses (a) that the diffusion of intracellular K⁺ into the sucrose would result in an increase in tissue impedance and (b) that a layer of trapped electrolyte under the oil film provides a shunt pathway are examined by computer analyses of a simple model.     

Quantitative Aspects of a Theory of Translocation

An analysis is made of two aspects of the behaviour of a proposedmechanism of translocation based on the mixing effect withina sieve-tube of streaming transcellular strands of protoplasmpassing through the sieve-pores and through a vacuole-like reservoirof sucrose solution in each sieve-element. First, the behaviour of the model is considered when it is transportinga solute (sucrose, for example) in the steady-state conditionof constant consumption and supply. It is found that the concentrationsof solute in the regions of the model are readily determinedat all points of the sieve-tube, and that the gradients of concentrationare all equal, the concentrations differing by constant amountsat all points. Transport is analogous to diffusive transferin that the steepness of the concentration gradient is proportionalto the rate of transfer and simple expressions are derived forthe net transport of solute and for the apparent diffusion coefficientin terms of the properties of the model. Transfer is found tobe rapid over a short path and slower over a longer path; thereis a maximum distance beyond which a given rate of transfercannot take place. Second, an analysis is made of the unsteady behaviour of themodel when changing concentrations of solute are introducedinto the system. The concept of strand diffusion time is introduced.This is the time taken for the concentration in the strand surroundedby an infinite reservoir to fall to I/e of its initial value.The results of this analysis are used to determine how radioactivesolute will become distributed when it is introduced into thesteady-state model. If the time over which the translocationof radioactive solute has proceeded is small compared with thestrand diffusion time, a wave-like profile of radioactivitywith distance is produced, and if large, the profile is of adiffusive kind; intermediate conditions give a profile displayingboth characteristics which is termed the transition type. Thesethree types of profile of radioactivity are exemplified by experimentsin different plants (Salix, Tropaeolum, and Helianthus), andthe experimental profiles are used to estimate (1) the apparentdiffusion coefficient of translocated sucrose in the sense ofMason and Maskell, and (2) the permeability constant of thestrand-reservoir boundary. The close correspondence betweenthese two sets of figures derived from the radioactive profileswith the experimental values obtained directly is regarded asstrong confirmation of the theory.     

Evidence for copurification of micronuclei in sucrose density gradient-enriched plasma membranes from cell lines

Sucrose density gradient-enriched membrane preparations and membrane fraction enrichment through affinity purification techniques are commonly used in proteomic analysis. However, published proteomic profiles characterized by the above methods show the presence of nuclear proteins in addition to membrane proteins. While shuttling of nuclear proteins across cellular compartments and their transient residency at membrane interfaces could explain some of these observations, the presence of nuclear proteins in proteomic profiles generated with crude and enriched membranes could be the result of nonspecific contamination of nuclear debris during cell fractionation procedures. We hypothesized that micronuclei arising from the genomic instability inherent to cancer cells may copurify with plasma membrane fractions on sucrose gradients. Using sucrose gradient-enriched plasma membranes from breast cancer cell lines derived from the MCF-7 cell line, we provide experimental evidence to indicate that micronuclei are present in fresh preparations of plasma membranes. The origin of these micronuclei was traced to budding of nuclei in intact cells. Furthermore, mass spectrometric analysis confirmed the presence of nuclear proteins as well as membrane and associated signaling proteins in sucrose gradient-enriched preparations.     

Exact solution of a model of diffusion in an infinite chain or monolayer of cells coupled by gap junctions.

Analytic solutions are found for an infinite chain of cells coupled by gap junctions under two initial conditions: (a) One inner cell initially filled uniformly to a fixed concentration and (b) inner cell maintained indefinitely at constant concentration. The solution can be extended by the product method (Carslaw and Jaeger. 1959. Conduction of Heat in Solids. Oxford University Press.) to monolayers. We can also incorporate leakage through the plasma membrane by the product method. We demonstrate the utility of these results by fitting diffusion data from the septate axon of earthworm and by plots of theoretical profiles from monolayers of cells. Use of these analytic solutions enables one to overcome the limitations of methods that lump the effects of cytoplasmic diffusion and junctional permeability into an effective diffusion coefficient.     

Some permeability properties of isolated rat liver cell mitochondria

The rates of penetration of various solutes into isolated rat liver mitochondria have been studied. Sodium, potassium, and sucrose were observed to enter the mitochondria until an equilibrium concentration was reached. The diffusion of these solutes, after the first few minutes, followed the predicted diffusion curve for solutes entering a particle with a rate-limiting membrane and instantaneous mixing in the interior. Reasons for deviations from the predicted equation during the first few minutes of diffusion are suggested. The data show that at pH 7.4 sodium and potassium enter more rapidly than sucrose. I¹³¹-labelled albumin was found to enter very slowly, if at all. Increasing the pH from 7.4 reduced the rate at which sodium ion penetrated the mitochondria. The rate of diffusion of sucrose into mitochondria was considerably slower than diffusion of sucrose into a sphere of water of the same size. Sodium ion was not found to be concentrated in vitro against an external concentration gradient as has been reported by other investigators. It is concluded that the rate of diffusion of solutes between the external medium and the interior of mitochondria is probably restricted and controlled by a mitochondrial membrane exhibiting passive permeability characteristics.     

Voltage-clamp of cut-end skeletal muscle fibre: a diffusion experiment

Membrane potential and current were studied in cut end fibres of frog skeletal muscle under current and voltage clamp conditions, by the double sucrose gap technique. Similar action potentials were recorded under current clamp conditions with either the microelectrode or the double sucrose gap techniques. Under voltage clamp conditions, the control of the membrane potential was maintained adequately. The early current was sensitive to both TTX and external Na concentration suggesting that the current was carried by Na ions. Sodium current (INa) was subsequently analysed using the Hodgkin-Huxley formulae. INa half-activation and inactivation occurred at -34 mV and -60 mV, respectively. Na-rich solution applied internally by diffusion through cut ends produced a reduction of INa associated with a shift of the sodium current reversal potential (VNa) towards more negative membrane potentials. This suggested that the sodium electromotive force was reduced by the increase in internal Na content of the fibre. Iodate applied externally changed neither the activation nor the inactivation time courses of INa, but reduced the peak current. Conversely, internally applied by diffusion from the cut end of skeletal muscle fibre, iodate slowed down the time course of INa inactivation and decreased the current peak. In conclusion, the double sucrose gap technique adapted to cut end frog skeletal muscle fibre allows a satisfactory analysis of INa.     

Quantitative Examination of Tissue Concentration Profiles Associated with Microdialysis

Spatial solute concentration profiles resulting from in vivo microdialysis were measured in rat caudate-putamen by quantitative autoradiography. Radiolabeled sucrose was included in the dialysate, and the tissue concentration profile measured after infusions of 14 min and 61.5 min in an acute preparation. In addition, the changes in sucrose extraction fraction over time were followed in vivo and in a simple in vitro system consisting of 0.5% agarose. These experimental results were then compared with mathematical simulations of microdialysis in vitro and in vivo. Simulations of in vitro microdialysis agreed well with experimental results. In vivo, the autoradiograms of the tissue concentration profiles showed clear evidence of substantial differences between 14 and 61.5 min, even though the change in extraction fraction was relatively small over that period. Comparison with simulated results showed that the model substantially underpredicted the observed extraction fraction and overall amount of sucrose in the tissue. A sensitivity analysis of the various model parameters suggested a tissue extracellular volume fraction of approximately 40% following probe implantation. We conclude that the injury from probe insertion initially causes disruption of the blood-brain barrier in the vicinity of the probe, and this disruption leads to an influx of water and plasma constituents, causing a vasogenic edema.     

Comparison of the polypeptide composition of Escherichia coli outer membranes prepared by two methods.

Escherichia coli outer membranes were prepared by centrifugation to equilibrium in sucrose gradients and then treated with Sarkosyl in the presence of ethylenediaminetetraacetate. The polypeptide profiles of the two outer membrane preparations were compared by two-dimensional polyacrylamide gel electrophoresis. The patterns obtained were not identical, and Sarkosyl removed several minor proteins from the outer membrane.     

Membranes of Rhodospirillum rubrum: isolation and physicochemical properties of membranes from aerobically grown cells.

Highly purified preparations of cytoplasmic and outer membrane were isolated from aerobically grown Rhodospirillum rubrum lysed by sequential treatment with lysozyme, ethylenediaminetetraacetate, and Brij 58. The membranes were resolved and separated from other cellular constitutents by a combination of velocity and isopyknic sedimentation in sucrose density gradients. On the basis of their appearance in electron micrographs and their protein profiles in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these preparations appear to be quite similar to those obtained from other gram-negative bacteria. The cytoplasmic membrane fraction contained the majority of the total membrane-bound succinic dehydrogenase activity and was 10-fold enriched in b- and c-type cytochrome with respect to the outer membrane. The latter fraction was characterized by a much greater carbohydrate content and the presence of arachidic acid, which is typical of R. rubrum lipopolysaccharide. Their protein fatty acid, and overall chemical compositions suggested that these preparations were freer from cross-contamination than those obtained from R. rubrum with currently available methods.     

ADPG formation by the ADP-specific cleavage of sucrose-reassessment of sucrose synthase.

The standardized enzyme coupling method for assaying sucrose synthase activities in the direction of sucrose cleavage was reexamined using enzyme preparations from cultured cells of sycamore (Acer pseudoplatanus L.) and spinach leaves (Spinacea oleracea). Both ATP and Tris, commonly utilized in assay systems to measure sucrose synthase, were found to inhibit non-competitively the ADPG-synthesizing activities of the enzyme. Upon substituting ATP by either GTP or UTP, and Tris by HEPES, we found that the sucrose synthase is capable of producing ADPG effectively, recognizing ADP as the principal substrate (Km = 5.3 microM (sycamore) and 16.8 microM (spinach]. The Vmax value for the synthesis of ADPG clearly surpasses the Vmax observed for the synthesis of UDPG by the enzyme. It was found that UDP is not inhibitory on the synthesis of ADPG by SS, which behaves allosterically with respect to the concentration level of sucrose.     

Electrical Transmission at the Nexus between Smooth Muscle Cells

The hypothesis that nexuses between cells are responsible for the core conductor properties of tissues was tested using smooth muscle preparations from the taenia coli of guinea pigs. Action potentials recorded from small diameter preparations across a sucrose gap change from monophasic to diphasic when a shunt resistor is connected across the gap. This indicates that transmission between smooth muscle cells is electrical, because the resistor only allows current to flow. Nexal fusion of cell membranes occurs especially where one cell sends a large bulbous projection into a neighbor. Hypertonic solutions rupture the nexuses between smooth muscle cells. Hypertonicity also increases the resistance of a bundle across the sucrose gap and blocks propagation of action potentials. Thus the structural and functional changes in smooth muscle due to hypertonicity correlate with the hypothesis.