In vitro evidence of propagation of superoxide dismutase-1 protein aggregation in canine degenerative myelopathy.

Canine degenerative myelopathy (DM) is a progressive and fatal neurodegenerative disorder that has been linked to mutations in the superoxide dismutase 1 (SOD1) gene. The accumulation of misfolded protein aggregates in spinal neurons and astrocytes is implicated as an important pathological process in DM; however, the mechanism of protein aggregate formation is largely unknown. In human neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), cell-to-cell propagation of disease-relevant proteins has been demonstrated. Therefore, in this study, propagation of aggregation-forming property of mutant SOD1 protein in DM in vitro was investigated. This study demonstrated that aggregates composed of canine wild type SOD1 protein were increased by co-transfection with canine mutant SOD1 (E40K SOD1), indicating intracellular propagation of SOD1 aggregates. Further, aggregated recombinant SOD1 proteins were released from the cells, taken up by other cells, and induced further aggregate formation of normally folded SOD1 proteins. These results suggest intercellular propagation of SOD1 aggregates. The hypothesis of cell-to-cell propagation of SOD1 aggregates proposed in this study may underly the progressive nature of DM pathology.

Selective modification of positive chemotaxis in the true slime mold Physarum polycephalum by ethylenediaminetetraacetic acid treatment.

The plasmodium of the true slime mold Physarum polycephalum was treated with EDTA or EGTA and the effect of the treatment on the chemotactic response was examined by measuring the chemotactic motive force with the double-chamber method. The results obtained were as follows: (1) The treatment of the plasmodium with 5 mM EDTA (pH 7.0, 20 min) did not give any significant effect on the protoplasmic streaming or motility. (2) The plasmodium treated with EDTA exhibited no chemotactic response to non-electrolyte attractants (D-glucose, D-galactose, D-mannose, and maltose) and negative chemotaxis to electrolyte attractants (cyclic AMP and NaH2PO4). (3) The EDTA treatment gave no effect on the chemotactic response to repellents (D-fructose, NaCl, CaCl2, MgCl2). (4) The EDTA-treated plasmodium exhibited changes in the membrane potential in response to both attractants and repellents as similar to the untreated plasmodium. (5) The treatment of the plasmodium with 5 mM EGTA (pH 7.0, 20 min) gave results similar to those obtained with the EDTA treatment. The results obtained suggested that EDTA (or EGTA) treatment did not affect the receptor sites but modified the transduction mechanism from reception into tactic movement.

Mitochondrial membrane potential estimated with the correction of probe binding.

Lipophilic ions are widely used as the probe for estimation of the membrane potential. It is suggested that the correction of the probe binding to the membrane and/or intracellular constituents is a problem to be solved in order to evaluate the membrane potential accurately. Previously, we proposed a method for the correction of the probe binding (Demura, M., Kamo, N. and Kobatake, Y. (1985) Biochim. Biophys. Acta 820, 207-215). In this paper, the method was applied to the determination of the membrane potential of intact mitochondria. The probes used constitute a homologous series of (Phe)3-P+-(CH2)n-CH3 (n = 0-4) and tetraphenylphosphonium (TPP+). Binding of these probes to de-energized mitochondria followed the Langmuir isotherm. However, values of parameters determined at high (50-800 microM) and low (under 20 microM) probe concentrations were different, suggesting the existence at least two, high- and low-affinity, binding sites. With extrapolation to the 'state of no binding', the membrane potential of intact mitochondria was estimated to be -147 mV (interior-negative) when they were energized by 5 mM succinate in medium consisting of 125 mM KCl, 10 mM MgCl2, 5 mM phosphate, 0.4 mM EDTA and 50 mM Tris-HCl (pH 7.5) at 25 degrees C. Parameters appearing in the equation for the correction of probe binding were determined with the use of this value of the membrane potential. The validity of the equation and the value of the parameters were revealed by the fact that after the correction, all probes used gave approximately the same value under the same conditions. We expanded the method so as to include the langmuir adsorption isotherm. When the modified equation is used, the estimated membrane potentials were less dependent on a probe concentration less than 10 microM.

Binding of lipophilic cations to the liposomal membrane: thermodynamic analysis.

Lipophilic ions are widely used as probes for measuring membrane potentials. Since binding of the probes to the membrane interferes with the accurate estimation of the membrane potential, it is necessary to clarify the characteristics of probe binding to membranes. The present paper deals with the binding of lipophilic cations to liposomes. The results can be summarized as follows: (1) The binding of triphenylmethylphosphonium, its homologues and tetraphenylphosphonium to liposomes of dipalmitoylphosphatidylcholine followed the Langmuir adsorption isotherm. (2) Spin-labeled lipophilic cations were synthesized and the binding to liposomes of egg phosphatidylcholine was examined. The binding also followed the Langmuir adsorption isotherm. The dissociation constant (the concentration giving half-maximal binding), K, was independent of the temperature, indicating that the binding is entropy-driven. (3) The binding was influenced by the fluidity of the membrane. Except in the case of triphenylmethylphosphonium (TPMP+), K and A (maximum amounts of binding) increased above the transition temperature. In other words, above the phase transition temperature the binding affinity is decreased, while maximum amounts of binding are increased for all phosphoniums used except TPMP+.

Pysicochemical studies of taste reception. V. Suppressive effect of salts on sugar response of the frog.

The tast responses of frog to various kinds of sugars were measured quantitatively by use of the glossopharyngeal nerve activity under an appropriate condition where the water response was completely suppressed. The concentration dependences of response of frog tongue to D-fructose, D-glucose, and sucrose were almost the same, D-galactose, however, elicited a much larger response in comparison with the other sugars in the whole range of concentrations examined. The sugar response was suppressed extensively by the presence of small amount of salts in the stimulating sugar solution. The suppressive effects of NaCl, KCl, MgCl2, MgSO4, and K4Fe(CN)6 were examined with a fixed concentration of sugar. The results obtained with these salts, added in various concentrations, fell on a single curve when the data were plotted against the ionic strength in the stimulating solution. The present results were consistent with the notion that the taste receptor potential for salts or acids is attributable to a change in the phase boundary potential at the membrane-solution interface as proposed in the previous papers of this series.

Selective electrode for dibenzyl dimethyl ammonium cation as indicator of the membrane potential in biological systems.

The electrode sensitive to dibenzyl dimethyl ammonium (DDA+), which is considered to be an indicator of the membrane potential, was constructed by using tetraphenyl borone (TPB-) embedded in dichloroethane. Rapid and Nernstian responses were exhibited against DDA+ solutions ranging between 10(-2) and 3 - 10(-6) M in concentration. High selectivity for DDA+ was observed in the presence of various inorganis salts, ADP, ATP, oxidizable substrates and sugars. The electrode developed here was used to measure the DDA+ uptake in Streptococcus faecalis and the results agreed with those reported by Harold, F.M. and Papineau, D. ((1972) J. Membrane Biol. 8, 27-44 and 45-62). While they determined the DDA+ concentration in the medium by measuring the absorbance of the filtrate treated with the ion-exchangers, the electrode can measure directly the DDA+ concentration in the bacterial suspension without any any pretreatment. It was also shown that the electrode can measure the DDA+ uptake in mitochondria during energization.

Physicochemical studies of taste reception. III. Interpretation of the water response in taste reception.

The model membrane composed of a Millipore filter paper and the total lipids from bovine tongue epithelium or phosphatidylcholine from egg yolk simulated well the water response of a living taste cell, The water response observed with the model membrane adapted to various salt solutions was interpreted in terms of changes in electric potential at the membrane-solution interface, i.e. the water response was attributed to the e.m.f. change produced by diffusion of the electrolytes dissolved in (or adsorbed on) the membrane surface into the bulk solution. The water response of the frog tongue was also investigated by measuring the neural response of the glossopharyngeal nerve. The results obtained were consistent with the mechanism proposed in the present paper. The response of the frog to Ca2+ was examined under the condition where the water response was suppressed, and it was concluded that the water response of the frog is different from the response to Ca2+.

Change in zeta potential and membrane potential of slime mold Physarum polycephalum in response to chemical stimuli.

Electrophoretic study of microplasmodia of the true slime mold Physarum polycephalum was carried out in the presence of various concentrations of inorganic salts, nucleotides and sugars, and the zeta potential at the surface of the plasmodia was determined from the electrophoretic mobilities. The membrane potential of the plasmodia was also measured under the same external conditions. It was shown that changes in the membrane potential induced by the chemical stimuli agreed approximately with those induced in the zeta potential in all cases examined. These results suggested that the phase boundary potential at the membrane-solution interface is mainly responsible for the membrane potential in the chemoreception of the slime mold.

Conversion of positive to negative chemotaxis by Ca2+ ionophore treatment in plasmodium of Physarum polycephalum.

The plasmodium of Physarum polycephalum was treated with Ca2+ ionophores A23187 or X-537A, and the effects on the chemotactic and contractile activities were studied by applying a double-chamber method and an optical method. (1) At low Ca2+ concentration, attractants (glucose, L-alanine) KH2PO4) remained as attractants and induced a relaxation in the plasmodial strand. Increase in the external Ca2+ level caused a contraction of plasmodium with the same stimuli, and hence converted the attractants to repellents. (2) This phenomenon was specific to Ca2+; Mg2+, Sr2+ or Ba2+ showed no appreciable effects. (3) Repellents (sucrose, KCl, Quinine) induced a contraction, which was not altered by the Ca2+ ionophore treatment. (4) Efflux of Ca2+, which was measured by chemiluminescence of aequorin, increased on glucose reception in the presence of ionophore.

Measurement of membrane potential in polymorphonuclear leukocytes and its changes during surface stimulation.

The membrane potential of guinea pig polymorphonuclear leukocytes has been assessed with two indirect probes, tetraphenylphosphonium (TPP+) and 3,3'-dipropylthiadicarbocyanine (disS-C3-(5)). The change in TPP+ concentration in the medium was measured with a TPP+-selective electrode. By monitoring differences in accumulation of TPP+ in media containing low and high potassium concentrations, a resting potential of -58.3 mV was calculated. This potential is composed of a diffusion potential due to the gradient of potassium, established by the Na+, K+ pump, and an electrogenic potential. The chemotactic peptide fMet-Leu-Phe elicits a rapid efflux of accumulated TPP+ (indicative of depolarization) followed by its reaccumulation (indicative of repolarization). In contrast, stimulation with concanavalin A results in a rapid and sustained depolarization without a subsequent repolarization. The results obtained with TPP+ and diS-C3-(5) were comparable. Such changes in membrane potential were observed in the absence of extracellular sodium, indicating that an inward movement of sodium is not responsible for the depolarization. Increasing potassium levels, which lead to membrane depolarization, had no effect on the oxidative metabolism in nonstimulated or in fMet-Leu-Phe-stimulated cells. Therefore, it seems unlikely that membrane depolarization per se is the immediate stimulus for the respiratory burst.

Selective enhancement and suppression of frog gustatory responses to amino acids.

Properties of the receptor sites for L-amino acids in taste cells of the bullfrog (Rana catesbeiana) were examined by measuring the neural activities of the glossopharyngeal nerve under various conditions. (a) The frogs responded to 12 amino acids, but the responses to the amino acids varied with individual frogs under natural conditions. The frog tongues, however, exhibited similar responses after an alkaline treatment that removes Ca2+ from the tissue. The variation in the responses under natural conditions was apparently due to the variation in the amount of Ca2+ bound to the receptor membrane. (b) The responses to hydrophilic L-amino acids (glycine, L-alanine, L-serine, L-threonine, L-cysteine, and L-proline) were of a tonic type, but those to hydrophobic L-amino acids (L-valine, L-leucine, L-isoleucine, L-methionine, L-phenylalanine, and L-tyrptophan) were usually composed of both phasic and tonic components. (c) The properties of the tonic component were quite different from those of the phasic component: the tonic component was largely enhanced by the alkaline treatment and suppressed by the acidic treatment that increases binding of Ca2+ to the tissue. Also, the tonic component was suppressed by the presence of low concentrations of salts, or the action of pronase E, whereas the phasic component was unchanged under these conditions. These properties of the phasic component were quite similar to those of the response to hydrophobic substances such as quinine. These results suggest that the hydrophilic L-amino acids stimulate receptor protein(s) and that the hydrophobic L-amino acids stimulate both the receptor protein and a receptor site similar to that for quinine. (d) On the basis of the suppression of the responses to amino acids by salts, the mechanism of generation of the receptor potential is discussed.

Effect of Ca2+, cyclic GMP, and cyclic AMP added to artificial solution perfusing lingual artery on frog gustatory nerve responses.

The lingual artery of the bullfrog was perfused with artificial solution and the effects of Ca2+, Ca-channel blockers (MnCl2 and verapamil), cGMP, and cAMP added to the perfusing solution of the gustatory nerve responses were examined. The responses to chemical stimuli of group 1 (CaCl2, NaCl, distilled water, D-galactose, and L-threonine) applied to the tongue surface were greatly decreased by a decrease in Ca2+ concentration in the perfusing solution, suppressed by the Ca-channel blockers, enhanced by cGMP, and suppressed by cAMP. The responses to chemical stimuli of group 2 (quinine hydrochloride, theophylline, ethanol, and HCl) were practically not affected by a decrease in Ca2+ concentration, the Ca-channel blockers, cGMP, and cAMP. The responses to the stimuli of group 1 seem to be induced by Ca influx into a taste cell that is triggered by depolarization and modulated by the cyclic nucleotides in a taste cell. The responses to group 2 seem to be induced without accompanying Ca influx.

Threshold phenomena in chemoreception and taxis in slime mold Physarum polycephalum.

The plasmodium of Physarum polycephalum reacts to various kinds of chemicals substances and moves towards or away from them. Threshold concentration of recognition of chemicals was examined in terms of membrane potential and of the averaged motive force of tactic movement by using a double-chamber method, i.e., a single plasmodium was placed between two compartments through a narrow ditch, and differences in membrane potential and in pressure between two compartments were measured. Results are summarized as follows: (a) By increasing the concentration of various substances in one compartment, the membrane potential started to change at a certain threshold concentration, C-th, for each chemical. Chemotactic movement of the plasmodium took place at the same threshold concentration. These results held both for attractants (glucose, galactose, phosphates, pyrophosphates, ATP, c-AMP, etc) and for repellents (various inorganic salts, sucrose, fructose, etc.). (b) The threshold concentration, Cth, for inorganic salts decreased remarkably with increase of the valences of cations, zeta, and was proportional to Z-6, I.E., THE Shultze-Hardy rule known in the field of colloid chemistry was found to be applicable. (c) The plasmodium distinguished the species of monovalent cations in the following order: H(Li(K(Na(Rb(Cs(NH-4 Plots of log Cth against the lyotropic number of anion fell on different straight lines for each monovalent cation species. (d) Plots of log Cth, against the reciprocal of the absolute tempe lines were almost the same and gave a value of 12 kcal/mol for the enthalpy change. These results suggest that the recognition of chemical substances appears as the result of a structural change of the membrane at the threshold point, and that the change in membrane structure is transmitted simultaneously to the motile system of the plasmodium.

Gustatory responses of eel palatine receptors to amino acids and carboxylic acids.

The gustatory receptors of the eel palate were found to be extremely sensitive to amino acids and carboxylic acids. The results obtained are as follows: (a) 11 amino acids which are among naturally occurring amino acids elicited responses in the palatine nerve, but 9 amino acids did not elicit a response even at a high concentration. The effect of D-amino acids was always much less than that of their corresponding L-isomers. There was no appreciable difference in the effectiveness of an alpha-amino acid (alpha-alanine) and beta-amino acid (beta-alanine). (b) The threshold concentrations of the most potent amino acids (arginine, glycine) were between 10(-8) and 10(-9) M. A linear relation between the magnitude of the response and log stimulus concentration held for a wide concentration range for all the amino acids examined. (c) The palatine receptors responded sensitively to various carboxylic acid solutions whose pH was adjusted to neutral. The threshold concentrations varied between 10(-4) and 10(-7) M. The magnitude of the response at 10(-2) M increased with an increase of carbon chain length. (d) The extent of cross-adaptation was examined with various combinations of amino acids. A variety of the response patterns showing complete cross-adaptation, no cross-adaptation, or synergetic interaction was observed. The synergetic interaction was also observed when one amino acid below its threshold concentration was added to the other amino acid below its threshold concentration was added to the other amino acid. No cross-adaptation was observed between amino acids and fatty acids. (e) The treatment of the palate with papain led to loss of the responses to arginine, glycine, and histidine without affecting those to proline and acetic acid. The treatment with pronase E eliminated selectively the response to proline. The possibility that the eel gustatory receptors are responsible for sensing food at a distance was discussed.

Taste transduction mechanism: similar effects of various modifications of gustatory receptors on neural responses to chemical and electrical stimulation in the frog.

Responses in the frog glossopharyngeal nerve induced by electrical stimulation of the tongue were compared with those induced by chemical stimuli under various conditions. (a) Anodal stimulation induced much larger responses than cathodal stimulation, and anodal stimulation of the tongue adapted to 5 mM MgCl2 produced much larger responses than stimulation with the tongue adapted to 10 mM NaCl at equal current intensities, as chemical stimulation with MgCl2 produced much larger responses than stimulation with NaCl at equal concentration. (b) The enhansive and suppressive effects of 8-anilino-1-naphthalenesulfonate, NiCl2, and uranyl acetate on the responses to anodal current were similar to those on the responses to chemical stimulation. (c) Anodal stimulation of the tongue adapted to 50 mM CaCl2 resulted in a large response, whereas application of 1 M CaCl2 to the tongue adapted to 50 mM CaCl2 produced only a small response. This, together with theoretical considerations, suggested that the accumulation of salts on the tongue surface is not the cause of the generation of the response to anodal current. (d) Cathodal current suppressed the responses induced by 1 mM CaCl2, 0.3 M ethanol, and distilled water. (e) The addition of EGTA or Ca-channel blockers (CdCl2 and verapamil) to the perfusing solution of the lingual artery reversibly suppressed both the responses to chemical stimulus (NaCl) and to anodal current with 10 mM NaCl. (f) We assume from the results obtained that electrical current from the microvillus membrane of a taste cell to the synaptic area supplied by anodal stimulation or induced by chemical stimulation activates the voltage-dependent Ca channel at the synaptic area.

Accumulation and aggregate formation of mutant superoxide dismutase 1 in canine degenerative myelopathy.

Canine degenerative myelopathy (DM) is an adult-onset progressive neurodegenerative disorder that has recently been linked to mutations in the superoxide dismutase 1 (SOD1) gene. We generated a polyclonal antibody against canine SOD1 to further characterize the mutant SOD1 protein and its involvement in DM pathogenesis. This antibody (SYN3554) was highly specific to canine SOD1 and had the ability to reveal distinct cytoplasmic aggregates in cultured cells expressing canine mutant SOD1 and also in the spinal neurons of symptomatic homozygotes. A similar staining pattern was observed in asymptomatic homozygotes. SOD1 aggregates were not detected in the spinal neurons of heterozygotes; the accumulation of SOD1 was also detected in the reactive astrocytes of homozygotes and heterozygotes to a similar extent. Our results support the hypothesis that the cytoplasmic accumulation and aggregate formation of the mutant SOD1 protein, especially in astrocytes, are closely associated with the pathogenesis of DM. Therefore, this disease is regarded as a spontaneous large-animal model of SOD1-mediated amyotrophic lateral sclerosis in humans.

Contraction rhythm in the plasmodium of Physarum polycephalum: dependence of the period on the amplitude, temperature and chemical environment.

The plasmodium of Physarum polycephalum exhibits a characteristic protoplasmic shuttle streaming and generates periodical tension. We determined the factors which govern the period of the contraction rhythm by measuring isometric tension of the plasmodial strand and the motive force of the protoplasmic streaming under a variety of conditions by changing the size of plasmodia, chemical composition of environment and surrounding temperatures. The results are: (1) The period of contraction rhythm, tau, increased linearly with the amplitude of oscillating tension, F, and was expressed by the following empirical equation when F was lower than a certain critical value, Fc, i.e., tau = aF+tau O. Above Fc, tau stayed at a constant level of tau S. There, a tau O and tau S are numerical constants which are independent of F. A similar relationship is valid for the amplitude of the motive force of the protoplasmic streaming, delta P. (2) Values of tau O were 1.0 min in air and 1.6 min in an aqueous medium and they were independent of temperature. (3) The Arrhenius plots of the parameter "a" exhibited different straight lines in air and in aqueous medium, from which the values of Q10 were determined to be 4.0 and 10, respectively. (4) Lowering of temperature decreased Fc, and eventually diminished it at Tc (= 15 degrees C). (5) The presence of glucose, CaCl2, MgCl2 and NaCl gave the identical tau-F relationship. Applications od D2O and ethanol slowed down the contraction rhythm, while that of KCl accelerated the rhythm. However, all these chemicals did not affect the tau O value. All results described above indicate that the contraction rhythm in the plasmodium is influenced by three components: a limit cycle which is independent of tension generation, a component which is strongly linked to the amplitude of the tension, F, and the part which is independent of the value of F. External factors appear to influence separately these three components of the contraction rhythm.

The effect of longitudinal tension on the amplitude of the contraction rhythm of plasmodial strands of Physarum polycephalum.

Plasmodial strands of Physarum polycephalum exhibit a characteristic contraction rhythm. The amplitude of the isotonic tension increased linearly with the cube root of relative stress. The period of the isometric tension increased linearly with the amplitude of the oscillation, but stayed constant above a critical value. The above empirical formulae were found applicable for all temperatures employed, 9 to 28 degrees C, but different temperature coefficients were observed below and above 15 to 16 degrees C.

Selective suppression of positive chemotaxis in Physarum polycephalum by treatment with rotenone or under anaerobic condition.

The chemotactic motive force of plasmodia of Physarum polycephalum was measured by the double-chamber method. The treatment of plasmodia with 0.1 mM rotenone did not affect the motility of the palsmodia but led to suppression of the chemotaxis toward all the attractants examined (glucose, galactose, c-AMP, KH2PO4). Rotenone treatment did not affect the chemotaxis against repellents (fructose, NaCl). Similar results were obtained when the chemotactic motive force was measured under an anaerobic condition (95% N2-5% CO2).

Membrane biophysics of chemoreception and taxis in the plasmodium of Physarum polycephalum.

The threshold phenomena observed in chemoreception and taxis of the plasmodium of Physarum polycephalum were analyzed on the basis of physical chemistry. Various physicochemical concepts and rules, e.g. the Schulze-Hardy rule, the lyotropic number and the hydrophobic interactions, were shown to be applicable reasonably well to the physiological functions in Physarum. It was stressed that the structural change of the surface membrane induced by reception of chemical stimuli plays a decisive role in recognition and sensitivity to the external stimuli as well as the appearance of tactic movement in the amoeboid motility of Physarum.