Adenine-nucleotide levels and metabolism-dependent membrane potential in cells of Nitellopsis obtusa Groves

The relationship between adenine-nucleotide levels and metabolism-dependent membrane potential was studied in cells of Nitellopsis obtusa. Effects of ADP and AMP in the presence of ATP on electrogenic pump activity were measured in the dark, using the continuous perfusion method. Both ADP and AMP acte as competitive inhibitors for ATP, the K_i value for either compound being about 0.4 mM. The role of ADP and AMP as regulating factors for the electrogenic pump was investigated under various metabolic conditions. Application of N₂ gas in the dark caused a significant membrane depolarization amounting to 90 mV, but cytoplasmic streaming and membrane excitability were not affected. Under anoxia, the ATP level decreased from 1.6 to 0.5 mM; ADP increased but only slightly, and AMP increased greatly. However, the time course of changes in the adenine nucleotides was not concurrent with that of the membrane-potential changes, thus, the adenine-nucleotide level changes cannot fully account for the N₂-elicited depolarization. Under light, although the membrane hyperpolarized, no significant changes in the adenine-nucleotide levels were observed. Therefore, the light-induced membrane hyperpolarization cannot be explained solely by changes in adenine-nucleotide levels.Abbreviations APW artificial pond water - E_m membrane potential - R_m membrane resistance     

Control of membrane potential and excitability ofChara cells with ATP and Mg2+

Summmary Electric characteristics of internodalChara australis cells, from which the tonoplast had been removed by vacuolar perfusion with media containing EGTA, were studied in relation to intracellular concentrations of ATP and Mg²⁺ using the ordinary microelectrode method and the open-vacuole method developed by Tazawa, Kikuyama and Nakagawa (1975.Plant Cell Physiol. 16:611). The concentration of ATP was decreased by introducing hexokinase and glucose into the cell and that of Mg²⁺ by introducing EDTA or CyDTA. The membrane potential decrease and the membrane resistance increase were both significant when the ATP or Mg²⁺ concentration was decreased. An ATP-dependent membrane potential was also found in other species of Characeae,Nitella axillaris andN. pulchella. Excitability of the membrane was also completely lost by reducing the ATP or Mg²⁺ concentration. Both membrane potential and excitability were recovered by introducing ATP or Mg²⁺ into ATP- or Mg²⁺-depleted cells.The time course of membrane potential recovery was followed by the open-vacuole method. Recovery began as soon as intracellular perfusion with medium containing ATP and Mg²⁺ was started. Reversible transition of the membrane potential between polarized and pepolarized levels by controlling the intracellular concentration of ATP or Mg²⁺ could be repeated many times by the open-vacuole method, when the excitability was suppressed by addition of Pb²⁺ to the external medium.The ineffectiveness of an ATP analog, AMP-PNP, and the synergism of ATP and Mg²⁺ in maintaining the membrane potential and excitability strongly suggest that ATP act via its hydrolysis by Mg²⁺-activated ATPase. The passive nature of the membrane, as judged from responses of the membrane potential to changes of the external K⁺ concentration, was not altered by lowering the ATP concentration in the cell. The mechanism of membrane potential generation dependent on ATP is discussed on the basic of an electrogenic ion pump. Involvement of the membrane potential generated by the ion pump in the action potential is also discussed.     

Demonstration of light-induced potential change in Chara cells lacking tonoplast

Chara cells without tonoplasts, prepared by replacing the cellsap with EGTA-containing media, showed essentially the samepattern of light-induced changes in membrane potential and membraneresistance as normal cells although the concentrations of ionsand ATP in the cytoplasm decreased considerably (1/3–1/10)after loss of the tonoplast. Removal of the tonoplast reducedthe rate of photosynthetic O₂ evolution to about 50% of thatof normal cells but did not affect the magnitude of light-inducedpotential change. Not a full but a certain level of electronflow seems necessary to activate the putative electrogenic H⁺-pump. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Japan. ² Present address: Niigata College of Pharmacy, Niigata 950-21,Japan. (Received September 4, 1978; )     

The micronucleus test with mouse peripheral blood on N-methyl-N'-nitro-N-nitrosoguanidine and mitomycin C.

The micronucleus test using mouse peripheral blood was conducted with N-methyl-N'-nitro-N-nitro-soguanidine (MNNG) and mitomycin C (MMC) as part of the 5th collaborative study supported by the Environmental Mutagen Society of Japan (CSGMT/MMS.JEMS). Male CD-1 mice were intraperitoneally injected once with 12.5-100 mg/kg of MMC. Peripheral blood was drawn at different intervals after treatment, placed on slides previously coated with acridine orange and the numbers of reticulocytes with micronuclei (MNRETs) were scored. The experiments indicated that the maximum effect of both MNNG and MMC was found about 48 h after treatment, and that the micronucleus test using peripheral blood is useful for the screening of chemicals throughout the experimental period in a single animal.     

Hydrogen exchange kinetics of nucleic acids: Double and triple helices with Hoogsteen-type basepairs

The kinetics of hydrogen-tritium exchange reaction have been followed by a Sephadex technique of a double-helical poly(ribo-2-methylthio-adenylic acid)·poly(ribouridylic acid) complex with the Hoogsteen-type basepair. Only one hydrogen in every 2-methylthio-adenine·uracil basepair has been found to exchange at a measurably slow rate, 0.023 s^?1 (at 0°C), which is, however, much greater than that for a double-helix with the Watson-Crick type A·U pair. The kinetics of hydrogen-tritium exchange were also examined by triple-helical poly(rU)·poly(rA)·poly(rU) which involves both the Watson-Crick and Hoogsteen basepairings. Here, three hydrogens in every U·A·U base triplet have been found to exchange at a relatively slow rate, 0.0116 s^?1 (at 0°C). The kinetics of hydrogen-deuterium exchange reactions of these polynucleotide helices have also been followed by a stopped-flow ultraviolet absorption spectrophotometry at various temperatures. On the basis of these experimental results, the mechanism of the hydrogen exchange reactions in these helical polynucleotides was discussed. In the triple helix, the rate-determining process of the slow exchange of the three (one uracil-imide and two adenine-amino) hydrogens is considered to be the opening of the Watson-Crick part of the U·A·U triplet. This opening is considered to take place only after the opening of the Hoogsteen part of the triplet.     

Fibrin membrane endowed with biological function VI. Immobilization of multienzymes of urea cycle

Four enzymes in urea cycle and inorganic pyrophosphatase were immobilized simultaneously into a matrix of fibrin fiber formed from fibrinogen by the concerted action of thrombin and blood coagulation Factor XIII. The immobilized multienzyme system not only had an ability to carry out urea cycle continuously at least over several hours, but also had a greatly improved efficiency over the corresponding soluble system.     

Blood oxygen dissociation curve of the frogsRana catesbeiana andRana brevipoda

Summary The blood O₂ dissociation curve was determined with a microphotometric reaction apparatus in two frog species,Rana catesbeiana andRana brevipoda, and formulated in terms of a modified Hill equation. The O₂ dissociation curve was in reasonable agreement with determinations of O₂ content, O₂ capacity, and pH (or ) in blood samples taken from various blood vessels and heart cavities. The blood O₂ affinity inRana catesbeiana (mean body weight 230 g, kept at 25°C) was low:P ₅₀=42 Torr (at 25°C and at ventricular blood pH=7.79). The blood of the smaller frog,Rana brevipoda (mean body weight 16 g), had even lower O₂ affinity,P ₅₀ being 52 Torr (at 25°C, pH=7.72). The decrease ofP ₅₀ with increasing body mass, resulting from comparison of these two frog species, is in accordance with literature data on mammals and birds.     

Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis

We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170?kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50?kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl₂, and HgCl₂ functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl₂ was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition.     

A clinical study of the efficacy of a single session of individual exercise for depressive patients,assessed by the change in saliva free cortisol level

Background

The efficacy of physical exercise as an augmentation to pharmacotherapy with antidepressants for depressive patients has been documented. However, to clarify the effectiveness of exercise in the treatment of depression, it is necessary to distinguish the effect of the exercise itself from the effect of group dynamics. Furthermore, an objective measurement for estimation of the effect is needed. Previous reports adopted a series of group exercises as the exercise intervention and mainly psychometric instruments for the measurement of effectiveness. Therefore, this clinical study was done to examine the effectiveness of a single session of individual exercise on depressive symptoms by assessing the change in saliva free cortisol level, which reflects hypothalamic-pituitary-adrenocortical axis function that is disturbed in depressive patients.

Method

Eighteen medicated patients, who met the DSM-IV-TR criteria for major depressive disorder, were examined for the change in saliva free cortisol levels and the change in subjective depressive symptoms before and after pedaling a bicycle ergometer for fifteen minutes. Within a month after the exercise session, participants conducted a non-exercise control session, which was sitting quietly at the same time of day as the exercise session.

Results

Depressed patients who participated in this study were in remission or in mild depressive state. However, they suffered chronic depression and had disturbed quality of life. The saliva free cortisol level and subjective depressive symptoms significantly decreased after the exercise session. Moreover, the changes in these variables were significantly, positively correlated. On the other hand, although the subjective depressive symptoms improved in the control session, the saliva free cortisol level did not change.

Conclusion

For the first time in depressive patients, we were able to show a decrease in the saliva free cortisol level due to physical exercise, accompanied by the improvement of subjective depressive symptoms. This identified a possible influence of exercise on the hypothalamic-pituitary-adrenal axis in depression.These results suggest the utility of assessing the effect of physical exercise by saliva free cortisol level in depressive patients who suffer from bio-psycho-social disability.