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.

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.

Changes in blood coagulation, platelet aggregation, and lipid metabolism in rats given lipids containing docosahexaenoic acid.

In order to identify an adequate intake level of docosahexaenoic acid (DHA), changes in various parameters related to health benefits were studied in rats fed on diets containing 10% test lipids at different n-3(DHA)/n-6 ratios for two weeks. An evaluation of the critical level of the dietary n-3/n-6 ratio which had a significant effect on the parameters of several tissues indicated that the response to the dietary ratios differed according to the parameter, the variation in ratio ranging approximately from 0.20 to 1.77 with either a positive or negative effect on the health benefit. These results suggest that a suitable intake level of DHA would be within this range. In view of safety, however, the critical level for the dietary n-3/n-6 ratio may be around 0.56, as shown by a detailed analysis on the lower limit level of the harmful parameters. We thus propose that the dietary intake of DHA should not be more than 0.56 in terms of the n-3/n-6 ratio.

Effects of octopine on the serum cholesterol level in rats.

The effects of dietary octopine, which is one of the major extractive component of marine molluscs, on the level of serum and liver cholesterol of rats fed with cholesterol-enriched or cholesterol-free diets were investigated. Dietary supplementation with 1.5% octopine in a cholesterol-enriched diet significantly decreased the serum total- and VLDL+LDL-cholesterol levels and by contrary increased the serum HDL-cholesterol level in rats. The same tendency was observed in the rats fed with 1.5% octopine in a cholesterol-free diet.

Earthquake - resistant capacity of reinforced concrete bridge columns retrofitted with carbon fibers

Reinforced concrete highway bridge columns in Japan have some seismic vulnerability, such as termination of main reinforcement at mid-height and poor transverse reinforcement. There are two dominant retrofitting methods, however, they does not sufficiently satisfy practical demands. A new retrofitting technique, that is, applying and/or wrapping carbon fiber sheets and strands onto the column's surface as longitudinal and transverse reinforcement has been developed. In order to investigate the structural performance of this technique and verify the design concept, static and cyclic loading tests with 1/3 scaled models are carried out. The tests results shows, that this method increase the flexural strength of main-bar-terminated part, and shear strength and ductility of the column itself. It is concluded that this technique is effective for improving earthquake-resistant capacity of existing reinforced concrete bridge columns (AU)

Photoreaction cycle of phoborhodopsin studied by low-temperature spectrophotometry.

The photochemical and subsequent thermal reactions of phoborhodopsin (pR490), which mediates the negative phototaxis (phobic reaction) of Halobacterium halobium, were investigated by low-temperature spectrophotometry. At room temperature, the absorption spectrum of pR490 displayed vibrational structure with a maximum at 490 nm and a shoulder at 460 nm, which were remarkably sharpened by cooling, resulting in the appearance of two well-separated peaks. On irradiation of pR490 at -170 degrees C, a photo-steady-state mixture composed of pR490 and two photoproducts, P520 and P480, was formed. P480 had an absorption maximum at 480 nm and thermally converted to pR490 above -160 degrees C, while P520 had an absorption maximum at 515 nm and thermally converted to P350, the next intermediate, above -60 degrees C. Above -30 degrees C, P350 was converted to P530, and then reverted to pR490. P520, P350, and P530 may correspond to K, M, and O intermediates of bacteriorhodopsin, respectively, on the basis of their absorption spectra, but the intermediates corresponding to L and N intermediates were not observed. On the basis of these results, a new scheme of the photoreaction cycle of pR490 was presented.

Influence of different types and levels of dietary lipids on liver microsomal mixed function oxidase system in rats.

It has been shown that dietary polyunsaturated fatty acids stimulate the liver microsomal mixed function oxidase system. The influence of different levels of dietary lard, soybean oil and sardine oil on the mixed function oxidase system was investigated in rats. The diet containing 5% sardine oil rich in eicosapentaenoic and docosahexaenoic acids stimulated the mixed function oxidase system, but the diet containing 5% lard in which lard consisted of 10.7% linolenic acid and 1.5% linolenic acid seemed unlikely to stimulate enough the mixed function oxidase system. On the other hand, no definite effects of large doses of dietary lipids, 25% in the diets, on the mixed function oxidase system were observed.

Plasma-polymerized membrane electrode for the determination of dextromethorphan and dimemorfan.

Ion-selective electrodes (ISEs) responsive to the antitussives dextromethorphan and dimemorfan were constructed by the fixation of an ion-exchanger, ammonium tetraphenylborate, on a Millipore membrane by means of a plasma-polymerization technique. The electrodes showed a Nernstian response over the range of 10(-5)-10(-2) M dextromethorphan and dimemorphan, and the working pH range was 5-7. The interference from common cations such as Na+, K+ and Ca2+ was negligible but some organic cations interfered weakly. The electrodes were applied successfully for the determination of the drugs in pharmaceutical preparations.

On the glutamate transport through cell envelope vesicles of Halobacterium halobium.

Glutamate uptake by envelope vesicles of Halobacterium halobium was measured. Previous authors showed that the glutamate uptake needs the illumination as well as Na+ gradient across the membrane. The latter is considered to be the driving force for the uptake. No satisfactory explanation for the necessity of the illumination has not been given. We found that in the absence of Cl- in the medium, only Na+ gradient was enough to induce the glutamate uptake, i.e. no illumination was needed. Glutamate uptake was measured with various strains of H. halobium. We found that the envelope vesicles prepared from strains containing no bacteriorhodopsin showed the glutamate uptake in the dark and in the presence of Cl- in the medium provided only that Na+ gradient is imposed.

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+.

Patterns in the distribution of intracellular ATP concentration in relation to coordination of amoeboid cell behavior in Physarum polycephalum.

The Physarum plasmodium reacts tactically to external stimuli. The cell behavior of this giant amoeboid cell was studied by analysing intracellular ATP concentration. The two-dimensional (2D) spatial distribution of ATP depended on cell shape: a polar pattern for a unidirectionally migrating plasmodium, a bowl shape for a circular plasmodium, a hump shape for an oval plasmodium, or a wavy pattern for plasmodia stimulated with blue light or confined in a small chamber, etc. Local external stimulation brought about new patterns of ATP distribution. The ATP concentrations around the stimulated frontal region were reduced by about a half stimulation with KCl (repellent) or casamino acids (attractant). In both cases, migration was inhibited. Migration velocity increased almost linearly with increasing concentration of intracellular ATP above the threshold (about 20 micrograms/mg protein). Under anaerobic conditions or at low temperatures, the intracellular ATP oscillated slowly with a periodicity of about 30 min. Pattern formations in the intracellular ATP concentration and amoeboid coordination are discussed in terms of coupled chemical oscillators in a self-organizing system.

Flash spectrophotometric identification of a fourth rhodopsin-like pigment in Halobacterium halobium.

A fourth retinal-containing pigment in Halobacterium halobium cell membrane was examined by flash spectrophotometry. The absorption maximum of this pigment was at about 480 nm. Flash light caused a photoreaction cycle with a half recovery time of about 300 ms at room temperature. The photoreaction cycle involved at least two photo-intermediates. The absorption maximum of the first one was at about 350 nm and that of the second was at around 530 nm. The spectral properties of this pigment and the content of the cells correlate with the sensitivity of photo-repellent response to the light around 480 nm. We suggest a name 'phoborhodopsin' for this new pigment.

Role of the valence of concanavalin A in the activation of guinea pig polymorphonuclear leukocytes.

Stimulation of polymorphonuclear leukocytes (PMN) by tetravalent concanavalin A (alpha-ConA) induces membrane depolarization preceding the onset of superoxide anion (O2-) production. Both divalent and monovalent ConA analogues were studied to evaluate the role of valence. Monovalent ConA (m-ConA) was inactive in stimulating O2- production and divalent derivatives were less active than native alpha-ConA. Similarly, membrane depolarization was dependent on the valency of ConA. m-ConA did not induce a marked change in membrane potential, whereas sustained depolarization occurred with multivalent ConA. The formation of multiple linked interactions between surface receptors may be an important early event in the activation of PMN by ConA.

Spatial and temporal organization of intracellular adenine nucleotides and cyclic nucleotides in relation to rhythmic motility in Physarum plasmodium.

Spatio-temporal organization of a migrating plasmodium was studied both by analysing intracellular concentrations of adenine and cyclic nucleotides and by applying image processing for recording oscillatory changes in thickness with use of microcomputers. ATP and ADP concentrations were about twice as high in the front as in the rear, while AMP distributed uniformly. On the other hand, cAMP and cGMP concentrations were several times higher in the rear than in the front, showing oscillations in between. The cAMP concentrations at the front oscillated with a phase advancing about one-third of the period with respect to the phase of the thickness oscillation, while cGMP concentration there varied only little. ATP concentration oscillated concomitantly with H+. A feedback control loop consisting of (ATP-H+)-cAMP-Ca2+ is proposed. The possible mechanism of rhythmic contractions involving mitochondria which may excrete pulses of Ca2+ and induce cell polarization is discussed.

Light and dark adaptation of halorhodopsin.

Dark incubation of envelope vesicles derived from a strain of Halobacterium halobium that lacks bacteriorhodopsin but contains halorhodopsin and a third rhodopsin-like pigment caused a decrease in the flash yield [the amplitude of a transient absorbance change of flash reactive component(s) by flash] of halorhodopsin but not the rhodopsin-like pigment. The flash yield decreased to reach a low steady level after incubation for about 4 days in the dark. The flash yield of halorhodopsin at any stage of dark incubation was increased by actinic illumination of the vesicles. The flash yield at 490 nm (absorbance increase) was found to be approximately proportional to that at 590 nm (absorbance decrease). These results indicate that halorhodopsin in the envelope vesicles has two forms, dark and light adapted, and that the halorhodopsin phototransient absorbing at 490 nm is originated from the light-adapted form. A difference spectrum between these two forms of halorhodopsin shows that the light-adapted halorhodopsin was red-shifted from the dark-adapted form. The light-induced membrane potential was measured by tetraphenylphosphonium uptake. The uptake by the dark-adapted vesicles was slower than that by the light-adapted vesicles, suggesting that only the light-adapted halorhodopsin has ion-transporting activity.

Evidence that the long-lifetime photointermediate of s-rhodopsin is a receptor for negative phototaxis in Halobacterium halobium.

The effect of blue background light on behavioral response of Halobacterium halobium to step-like stimulation with green-orange attractant light was examined. The results strongly support the previously proposed hypothesis that a long-lifetime photointermediate of s-rhodopsin is the photoreceptor for repellent light: the step-like increase in green-orange light was convertible from attractant stimulus to repellent one, when the cells were constantly illuminated with blue light. No difference of the threshold intensity of the blue background light was observed between the mutant strain that lacks both bacteriorhodopsin and halorhodopsin and the wild type strain, suggesting that the two light-driven ion pumps are not participant in sensing attractant light.

Oscillations in cell shape and size during locomotion and in contractile activities of Physarum polycephalum, Dictyostelium discoideum, Amoeba proteus and macrophages.

Changes in cell shape and size were measured during locomotion, together with the motive force of the protoplasmic streaming, in various amoeboid cells in different stages of their life cycle, and under various environmental conditions. The variations in these measurements with time were examined by Fourier spectral analysis. Notwithstanding a change in cell type in the life cycle of P. polycephalum, myxamoebae and tiny plasmodia showed a similar time pattern of locomotion, exhibiting oscillations having a mixture of several periods. A regular oscillation with protoplasmic streaming appeared in the plasmodium only above a critical cell size. D. discoideum amoebae oscillated with two periods of a few minutes in preaggregation stage, but with a period of 10 min in aggregation stage, the latter being induced by cAMP. Macrophages and A. proteus also oscillated with periods of a few minutes. Periods of all these oscillations were prolonged severalfold by respiratory inhibition with NaCN, but were unaffected by glycolytic inhibition with 2-deoxyglucose. Cell fragments of A. proteus containing fewer granules oscillated more slowly and with a larger amplitude than those containing more granules. Among the granules, the nucleus was excluded as a possible modifier of the oscillation. The oscillation in Physarum plasmodium was reversibly suppressed by combining respiratory and ATPase inhibitions in mitochondria with NaCN and oligomycin, intracellular ATP concentration being kept at an appropriate level. The present results show that amoeboid motility, as well as cell shape, is oscillatory and that mitochondria are involved in time keeping.

Negative phototaxis from blue light and the role of third rhodopsinlike pigment in halobacterium cutirubrum.

Wild-type cells of Halobacterium cutirubrum show phototaxis. In negative phototaxis the cells are repelled by blue-near ultraviolet light, and in positive phototaxis the cells are attracted to green-red light. The extent of the responses are measured by monitoring the changes in the reversal frequency of the swimming direction of cells using a computer-linked automated method as described previously (Takahashi, T., and Y. Kobatake, 1982, Cell. Struct. Funct., 7:183-192). When the intensity of the background light (illumination for the observation) was dramatically reduced, the sensitivity of the cells to the repellent light decreased markedly. This result has been previously explained by Bogomolni and Spudich (1982, Proc. Natl. Acad. Sci. USA, 79:6250-6254), who proposed that the photoreceptor for negative phototaxis is the long-lifetime intermediate in the photocycle of slow-rhodospin. The behavioral response in the negative phototaxis is dependent upon the intensity of the actinic light and the background light. This agrees quantitatively with our model based on the aforementioned hypothesis.