Experimental and functional changes in transmembrane potential and zeta potential of single cultured cells

Experiments were performed on single cells to investigate the relations between the total bioelectrical potential difference (PD) across the cell membrane (so-called transmembrane potential) and the net negative surface charge of the cell (zeta potential). The experiments were carried out on FL-cells, leucocytes and ovarian tumour cells. The PD was measured electrophysiologically by means of intracellular glass microelectrodes; the surface charge or the zeta potential was determined using cell electrophoresis. Both measuring methods are critically discussed.Under different conditions (hypothermia, hyperthermia, mitotic blocking agent, cell cycle), the transmembrane potential and zeta potential showed changes in an identical direction and often the response of transmembrane potential was found to be quicker and more intensive than that of the zeta potential. In other experiments (e.g. changing the extracellular Cl^? ion concentration) the reactions of both potentials showed no coincidence. Depending on the type of functionally or experimentally borne changes on the cytoplasmatic membrane, either both potentials or only one of them may be altered.     

Dynamic property of membrane formation in a protoplasmic droplet of Nitella

A theory is presented to explain the dynamic characteristics of an electric potential and the resistance of a surface membrane during the formation of a protoplasmic droplet isolated from Nitella. Basic equations are coupled ones for describing ion concentrations near the surface of the droplet, active and passive ion fluxes on the surface, and kinetics of membrane-constituting molecules diffusing from the inside of the protoplasm. The present results give a good explanation of the observed kinetics of electric properties throughout the formative process of surface membranes after the ion concentrations are replaced by lower ones. The results can also explain well the observed data on the steady state. Oscillatory changes in the membrane potential induced by ions strongly adsorbed on the surface membrane are discussed in relation to growth and regeneration phenomena in biological systems such as bean roots and Acetabularia.     

Oscillations bioélectriques dans des membranes de Nitella

The results of synchronous registering of bioelectrical potentials upon some cell phases under definite environemental conditons can be used more universally in studies on the nature of bioelectrical appearances in living plant cells. Determinations were made on different functional dependence of the bioelectrical potential of the outside cell membrane, the bioelectrical potential of cytoplasm and the bioelectrical potential of vacuola under the same environmental conditions. Different properties of the cell structures manifest unequal characteristics in rhytmical fluctuation of bioelectrical potentials; unsynchronous and synchronous rhytmical fluetuations in the bioelectrical potential of cytoplasm and the outside cell membrane might occur. The amplitudes of the cytoplasm biometrical potential and the corresponding amplitude of biometrical potential in the outside cell membrane range up to 70 mV and 15 mV, respectively.     

Shifts in steady potential levels and changes in the cerebral bioelectrochemical potential during orientation and conditioned reflexes in rabbits]

The shifts of the steady potential level (SPL), recorded with non-polarized electrodes, and the changes in bioelectrochemical potential (BEChPs), recorded with platinum electrodes, were led from the rabbit brain surface in chronic experiments. The stimuli, that were new for the animal, caused only SPL shifts (0.1-0.3 mv), BEChPs showing no changes. BEChPs changed (by tenths to several millivolts) only in the process of the conditioned reflex formation, during the pairing of the conditioned and reinforcing stimuli, during which the shifts of SPL were also observed (up to 0.5-1 mv), different from the ones during the orienting reflex. Simultaneous recording of the SPL shifts and the changes in BEChPs showed that these phenomena are external manifestations of independent processes. It is suggested that the brain activity involved in the perception and the analysis of the informational value of a new stimulus, is connected with bioelectrical processes, rather than with the metabolic ones. During the formation of the defensive conditioned reflex, along with the enhancing of the activity connected with bioelectrical processes, other type of activity appears which is accompanied with considerable metabolic shifts.     

DISSIMILARITY OF INNER AND OUTER PROTOPLASMIC SURFACES IN VALONIA

The protoplasm of Valonia macrophysa forms a delicate layer, only a few microns in thickness, which contains numerous chloroplasts and nuclei. The outer surface is in contact with the cell wall, the inner with the vacuolar sap. As far as microscopic observation goes, these two surfaces seem alike; but measurements of potential difference indicate that they are decidedly different. We find that the chain sap | protoplasm | sap gives about 14.5 millivolts, the inner surface being positive to the outer. In order to explain this we may assume that the protoplasm consists of layers, the outer surface, X, differing from the inner surface, Y, and from the body of the protoplasm, W. We should then have the unsymmetrical chain sap | X | W | Y | sap which could produce an electromotive force. If the two surfaces of such a very thin layer of protoplasm can be different, it is of fundamental significance for the theory of the nature of living matter.     

Potentiometric determinations in the protoplasm and cell-sap of Nitella

Summary A method is described for the construction and use of a non-polarizable microelectrode which is protected within a quartz electrode chamber. Thereby the electrode may be saturated with hydrogen or any other gas or liquid.Using it as a hydrogen electrode, potentiometric measurements were made in the protoplasm and cell-sap ofNitella.The protoplasm and cell-sap behaved very differently toward the hydrogen electrode.The protoplasm at once produced a potential of between + ·093 and + ·030 volts with respect to hydrogen zero. These values which obviously cannot represent the concentration of hydrogen ions within the protoplasm, are interpreted as indicating rather its oxidation-reduction potential.Potentiometric readings for the cell-sap gave pH values of 5·47 to 6·16. The latter value is regarded as more reliable.     

Behavior of protoplasm for survival in injured cells ofValonia ventricosa: involvement of turgor pressure

Summary By injuring cells ofValonia ventricosa, one of two survival strategies — wound-healing and protoplast formation — is induced. The present study revealed that turgor pressure, as well as Ca²⁺ in bathing medium, is involved in the choice between these survival strategies. On the process of wound-healing, turgor pressure is recovered in the presence of both the wound plug, which closes the wound immediately after an injury, and the aggregation of protoplasm around the wound, which serves to protect the inflow of outer medium into the protoplasm layer and also to strengthen the wound plug. When the size of the wound is more than 150 m in diameter, the protoplasmic aggregate strengthen the wound plug incompletely and, as a result, wound-healing is unsuccessful. In this case, the ejection of vacuolar sap is repeated, due to partial restoration of turgor pressure. In each ejection, the wound plug is blown off, together with the aggregated protoplasm and, after several ejections are repeated, the cell is unable to heal the wound because of a lack of protoplasm around the wound. Continuous depression of turgor pressure, during the repeat of the unsuccessful wound-healing, induces disorganization of the protoplasm layer. Under these conditions, the centrifugal propagation of protoplasmic aggregation, followed by the protoplasts formation, takes place easily. Effects of turgor pressure and Ca²⁺ in the bathing medium upon the wound healing is discussed and the cytoplasmic behavior for survival of wounded cells is presented schematically.     

An ultra simple model of protoplasm to test the theory of its long-range coherence and control so far tested (and affirmed) mostly on intact cell(s)

According to the association-induction hypothesis, the core of living phenomena lies in the long-range, one-on-many connectedness among all three major components of living protoplasm: protein, water and K+ (and the controlling agents, called the cardinal adsorbents.) This article describes simple experimental models that could cogently test the theory of this connectedness and its control by drugs and other cardinal adsorbents.     

NATURE OF THE ACTION CURRENT IN NITELLA : III. SOME ADDITIONAL FEATURES

Several forms of the action curve are described which might be accounted for on the ground that the outer protoplasmic surface shows no rapid electrical change. This may be due to the fact that the longitudinal flow of the outgoing current of action is in the protoplasm instead of in the cellulose wall. Hence the action curve has a short period with a single peak which does not reach zero. On this basis we can estimate the P.D. across the inner and outer protoplasmic surfaces separately. These P.D.''s can vary independently. In many cases there are successive action currents with incomplete recovery (with an increase or decrease or no change of magnitude). Some of the records resemble those obtained with nerve (including bursts of action currents and after-positivity).     

THE VARIATION OF ELECTRICAL RESISTANCE WITH APPLIED POTENTIAL : III. IMPALED VALONIA VENTRICOSA

Electrical resistance and polarization were measured during the passage of direct current across a single layer of protoplasm in the cells of Valonia ventricosa impaled upon capillaries. These were correlated with five stages of the P.D. existing naturally across the protoplasm, as follows: 1. A stage of shock after impalement, when the P.D. drops from 5 mv. to zero and then slowly recovers. There is very little effective resistance in the protoplasm, and polarization is slight. 2. The stage of recovery and normal P.D., with values from 8 to 25 mv. (inside positive). The average is 15 mv. At first there is little or no polarization when small potentials are applied in either direction across the protoplasm, nor when very large currents pass outward (from sap to sea water). But when the positive current passes inward there is a sudden response at a critical applied potential ranging from 0.5 to 2.0 volts. The resistance then apparently rises as much as 10,000 ohms in some cases, and the rise occurs more quickly in succeeding applications after the first. When the potential is removed there is a back E.M.F. displayed. Later there is also an effect of such inward currents which persists into the first succeeding outward flow, causing a brief polarization at the first application of the reverse potential. Still later this polarization occurs at every exposure, and at increasingly lower values of applied potentials. Finally there is a "constant" state reached in which the polarization occurs with currents of either direction, and the apparent resistance is nearly uniform over a considerable range of applied potential. 3. A state of increased P.D.; to 100 mv. (inside positive) in artificial sap; and to 35 or 40 mv. in dilute sea water or 0.6 M MgSO₄. The polarization response and apparent resistance are at first about as in sea water, but later decrease. 4. A reversed P.D., to 50 mv. (outside positive) produced by a variety of causes, especially by dilute sea water, and also by large flows of current in either direction. This stage is temporary and the cells promptly recover from it. While it persists the polarization appears to be much greater to outward currents than to inward. This can largely be ascribed to the reduction of the reversed P.D. 5. Disappearance of P.D. caused by death, and various toxic agents. The resistance and polarization of the protoplasm are negligible. The back E.M.F. of polarization is shown to account largely for the apparent resistance of the protoplasm. Its calculation from the observed resistance rises gives values up to 150 mv. in the early stages of recovery, and later values of 50 to 75 mv. in the "constant" state. These are compared with the back E.M.F. similarly calculated from the apparent resistance of intact cells. The electrical capacitance of the protoplasm is shown by the time curves to be of the order of 1 microfarad per cm.² of surface.     

Apparent violations of the all-or-none law in relation to potassium in the protoplasm

The protoplasm of Nitella forms a thin layer surrounding a large central vacuole filled with sap. At the inner surface of the protoplasm is a non-aqueous layer called Y and at the outer surface is a similar layer called X. At each of these layers there is a potential due to the diffusion of KCl in contact with the layer. We thus have P = P_X + P_Y in which P is the total potential, P_X is the potential at X, and P_Y the potential at Y. We assume that when stimulation occurs P_Y disappears and P_X remains unaltered. The loss of part of the potential therefore involves no violation of the all-or-none law since the Y layer loses all of its potential and the X layer loses nothing. If the concentration of KCl in the external solution and in the sap is known the concentration in the protoplasm can be calculated at each stimulation by measuring the height of the spike.     

The apparent viscosity of the protoplasm of subepidermal stem basis cells of Pisum sativum. Relation to aging, drought tolerance and water stress

Plants of Pisum sativum L. cv. Alaska wilt resistant were subjected to two different water stress regimes under controlled environment conditions: watering was stopped either on the 7th day (early stress) or on the 21st day (late stress) after planting. Plants under the early stress regime developed drought tolerance (adapted), while those under late stress did not. The apparent viscosity of the protoplasm of subepidermal stem basis cells was analyzed by the centrifugation and plasmolysis form method during the entire growth period.
The apparent viscosity of the subepidermal stem basis cells changed with plant age and was highest in 3-week-old plants. In controls the relation of apparent viscosity to age was the same when measured under full turgor and in relaxed state. Under early stress condition, however, the pattern of the viscosity changes with plant age was significantly different for turgescent and relaxed cells. In four week old plants, a higher apparent viscosity was measured in relaxed adapted cells than in relaxed control cells. It is suggested that the higher apparent viscosity is the result of a delayed cell aging.
Apparent viscosity was inversely proportional to soil moisture content and the osmotic potential of the cell sap for the cells of late stress plants, whereas no clear relation was found for the cells of early stress plants. This difference may indicate two mechanisms of viscosity changes: 1) osmotic dehydration of the protoplasm under water stress (passive viscosity change), 2) changes in the amount, hydration or architecture of macromolecules present in the cytoplasm (active viscosity change). Whereas differences in the apparent viscosity between control and stressed cells may not be the cause of drought tolerance, they seem to indicate the development of drought tolerance. Water stress history and plant age were the most critical factors controlling the apparent viscosity changes observed.     

A new method of breaking down the electroencephalogram into a system of oscillations that allows for the analysis of EEG phenomena of different durations

With the purpose of levelling the restrictions appearing at the EEG correlative-spectral analysis, particularly of short (1-2 sec) realizations, as a result of Fourier transform, a new algorithm is elaborated of decomposition and analysis of bioelectrical activity. The created algorithm of complete EEG reduction to the list of oscillations singled out has been tested by a corresponding program. It provides for the measurement of parameters of concrete characteristics of the curve, and the adequacy of decomposition is ascertained by the exact restoration from the list of oscillations of the initial EEG. The method shows good sensitivity to the revealing of transient EEG phenomena and also regional specificity of the cortical bioelectrical activity.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS : II. THE EFFECTS OF POTASSIUM ON TWO SPECIES WITH DIFFERENT SAPS

The potential difference across the protoplasm of impaled cells of two American species of Halicystis is compared. The mean value for H. Osterhoutii is 68.4 mv.; that for H. ovalis is 79.7 mv., the sea water being positive to the sap in both. The higher potential of H. ovalis is apparently due to the higher concentration of KCl (0.3 M) in its vacuolar sap. When the KCl content of H. Osterhoutii sap (normally 0.01 M or less) is experimentally raised to 0.3 M, the potential rises to values about equal to those in H. ovalis. The external application of solutions high in potassium temporarily lowers the potential of both, probably by the high mobility of K⁺ ions. But a large potential is soon regained, representing the characteristic potential of the protoplasm. This is about 20 mv. lower than in sea water. The accumulation of KCl in the sap of H. ovalis is apparently not due to the higher mobility of K⁺ ion in its protoplasm, since the electrical effects of potassium are practically identical in H. Osterhoutii, where KCl is not accumulated.     

Coumarin-induced disturbances of morphological development and cell wall formation in Trichophyton mentagrophytes.

When Trichophyton mentagrophytes thalli are placed on a medium containing 300 microgram/ml of coumarin, their growth rate is drastically reduced and the newly formed mycelium consists of curled and branched hyphae showing subapical bulges and swollen tips. Under the electron microscope, the most relevant abnormalities concern the cell walls which are often thickened by aberrantly shaped zones that are usually smeared unevenly over the surface of the "primary" wall instead of being deposited in discrete ridges. An ultracytochemical analysis suggests that the irregular thickenings are formed of chitin. Arguments suitable to explain the phenomena observed are discussed.     

Localization of crystals in diseased oats treated with uranyl acetate

Uranyl acetate, a suppressor of victorin-induced electrolyte leakage in oat leaves when applied together with, or before, victorin, also suppressed victorin-induced changes in ultra-structure. Uranyl crystallized in cell walls and near the plasmalemma of vascular cells, but was excluded from the protoplasm. Fewer crystals occurred near the plasmalemma when leaves were allowed to take up uranyl and victorin simultaneously than when uranyl alone was absorbed, but deposition in cell walls was similar in the two treatments. No differences in crystal distribution were found in uranyl-treated leaves which subsequently took up either water or victorin. The most striking effect of prolonged exposure to uranyl was increased vesicular activity in the protoplasm, formation of complex concentric membranes, and tonoplast damage. Following victorin treatment, uranyl post-treatment was ineffective in suppressing electrolyte leakage or preserving normal cellular ultrastructure. More severe ultrastructural damage was found in victorintreated leaves after uranyl post-treatments than after post-treatment with water, a result of victorin-induced damage which facilitates uranyl entry into the protoplasm.     

Rhythm assimilation in the isolated canine heart under isovolumic conditions

Isolated canine heart has an expressed ability for autoregulation of bioelectrical and contractile functions irrespective of the neurohumoral factors influence on the work of the heart, and Frank-Starling law. Under the change of stimulation frequency, the autoregulation of heart functions is carried out as rhythm assimilation at organ (cell) level. The heart has a higher ability to bioelectrical rhythm assimilation rather than the mechanical rhythm assimilation. Incomplete rhythm assimilation is characterised by the alternation of contractions. The "Everything or nothing" law has no applicability to the work of the heart.     

ELECTROPHYSIOLOGICAL STUDIES ON NITELLA CELLS, WITH SPECIAL REFERENCE TO THE ELECTRIC RESISTANCE

Electrical properties of Nitella (Tolypella) cells were studied,with special reference to their electric resistance The resistanceof the protoplasm, as well as the potential of the inside ofthe cell, was measured by inserting capillary electrode intothe cell, which had been sealed with vaseline externally atthe middle so that the current across the protoplasm could bereadily determined. The characteristics of the rectification and impedance weremeasured The electric current was found to flow across the protoplasmiclayer more easily from the sap to the external water than inthe reverse direction. The resistance and the capacitance ofthe cell varied with the a.c frequencies used for the measurement Simultaneous measurements were made of the action potential,the action current, and the resistance change of the protoplasmin order to determine the relationship between them An intimateparallelism between the time courses of these three phenomenawas discovered. An attempt was made to interpret the resultsby taking into account the dissociation of ions in the protoplasm. ¹Present address: Japan Atomic Energy Research Institute, Tokai,Ibaraki Prefecture. (Received May 8, 1962; )     

灵芝S3发酵培养基的优化及其对镰刀菌的抑制

以发酵液对镰刀菌孢子萌发的抑制率为指标,通过单因素试验,研究不同碳源、氮源、生长因子对灵芝S3发酵液抑菌活性的影响。运用响应面法,对各营养组分的含量进行优化。优化后碳源、氮源、生长因子含量分别为：乳糖3.04%,蛋白胨0.28%,VB1 0.0047%,抑菌率为91.71%,比基础发酵培养基增加了42.15%。发酵液作用12h后,光学显微镜下镰刀菌菌丝出现膨大和消融等畸变,并出现典型的“念珠状”形态;透射电镜显示镰刀菌孢子内出现大量空泡、原生质收缩,说明灵芝S3发酵液中的活性物质可有效抑制菌丝生长及孢子萌发。