Studies on the physical state of water in living cells and model systems. X. The dependence of the equilibrium distribution coefficient of a solute in polarized water on the molecular weights of the solute: experimental confirmation of the "size rule" in model studies

The equilibrium distribution of 14 sugars, sugar alcohols, and other nonelectrolytes in solutions of polyethylene oxide (PEO) and of native and alkali-denatured bovine hemoglobin were studied over wide concentration ranges. The results show that the equilibrium concentrations of all the solutes studies are rectilinearly related to their external concentrations. This straight-line relationship demonstrates the existence of these solutes entirely or almost entirely in the aqueous phase of these systems. Therefore the slope of each of these straight lines equals the equilibrium distribution coefficient or q-value of the solute involved. In general, the q-values decrease with increasing molecule weights (M.W.) of the solutes in 15% solutions of PEO, 20% solutions of alkali-denatured hemoglobin (and in 18% gelatin) but not in 39% solution of native hemoglobin. In solutions of PEO, of alkali-denatured hemoglobin studied (and of gelatin) a fraction of the water (20% to 30%) appears to have solvency similar to that of normal liquid water. The experimental findings of M.W.-dependent solute exclusion were discussed in the light of four alternative theories that have been offered to explain this type of phenomena. Among these four theories only the polarized multilayer theory agrees with most, if not all the facts known.     

A unitary cause for the exclusion of Na+ and other solutes from living cells, suggested by effluxes of Na+, D-arabinose, and sucrose from normal, dying, and dead muscles

1. The effluxes of labeled Na+, D-arabinose, and sucrose from normal muscle and muscle poisoned with low concentrations of iodoacetate were studied. The procedure involved repeated loading with isotope, followed by washing of the same muscle while still normal and at different states of dying. 2. The rates of Na+ efflux in both the fast and slow fraction remained either quite constant or showed some unpredictable, minor fluctuations. This was true for both Na+ and the two sugars studied, confirming earlier conclusions that the steady levels of these solutes were not maintained by pumps. 3. In all cases studied, the efflux curves showed at least two fractions. It is the fast-exchanging fraction that steadily and consistently increased in magnitude as the muscles were dying, until finally the concentration of solute in this fraction reached and sometimes surpassed the labeled solute concentrations in the original labeled solutions in which the muscles were equilibrated. The slow fractions showed only a transient increase or none at all. These observations show that it is the fast fraction that represents solute dissolved in cell water and rate-limited by passage through the cell surface and that the partial exclusion of Na+ and the sugars have a unitary cause--a reduced solubility in the cell water which in the presence of ATP exists in the state of polarized multilayers.     

Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions and have low toxicity to embryos and oocytes

A systematic approach was taken to assess the vitrification properties of ethylene glycol-based solutions supplemented with carbohydrates. Solutions were prepared by weight (gravimetrically) using ethylene glycol as the cryoprotectant, 0.9% NaCl in water, and six different sugars: d-glucose, d(-)-fructose, d-sorbitol, sucrose, d(+)-trehalose, and raffinose. Sugars were added on a molal basis (0. 1, 0.5, and 1 m). Characteristics of the solutions were measured during warming by differential scanning calorimetry using a cooling rate of 100 degrees C/min and a warming rate of 10 degrees C/min. In the absence of carbohydrates a 59 wt% EG-saline solution formed a stable glass. When EG was replaced by an equimolal concentration of glucose, fructose, or sorbitol (monosaccharides) at 0.1, 0.5, or 1.0 m there was no change in the total solute concentration at which vitrification occurred, but the glass transition (Tg) occurred at a higher temperature than in EG-saline alone. When EG was replaced by an equimolal concentration of sucrose or trehalose (disaccharides) both the Tg and the lowest total solute concentration required for vitrification became progressively higher as the molecular weight, or the ratio of sugar to EG in the solutions, increased. At the highest tested disaccharide concentration (1 m) vitrification was achieved at a total solute concentration of 65 wt% (sucrose) and 67 wt% (trehalose). The polysaccharide raffinose significantly modified the vitrification properties of ethylene glycol solutions. When 0.5 or 0.1 m raffinose replaced EG on an equimolal basis the glass transition point was raised more than with either the monosaccharides or the disaccharides. Raffinose allowed vitrification at a total solute concentration of 67 wt% (0.5 m) and 63 wt% (0.1 m). The maturation of immature mouse oocytes, and the development of embryos in media containing 5-7 mM of any sugar was comparable to controls, indicating that they are not toxic. Exposure of freshly collected GV or MII oocytes to sugar concentrations between 0.5 and 1.0 M, for up to 10 min had no significant effect on the proportion which subsequently formed two cells. We conclude that added sugars do contribute to a solutions overall vitrification properties, and their properties should be taken into consideration when vitrification solutions are being designed or modified.     

Studies on the physical state of water in living cells and model systems. XI. The equilibrium distribution coefficients of pentoses in muscle cell water: their dependence primarily on the molecular weights of the pentoses and lesser dependence on their stereospecificity

Studies of the metabolism of four pentoses (D-, and L-arabinose; D-, and L-xylose) in frog muscle at 0 degree C revealed that all are metabolized at extremely slow rates. As a result, the metabolic degradation of these pentoses does not significantly affect their equilibrium distribution in muscle cells at this temperature at least. Of the four stereoisomers, three (L-xylose, D-arabinose, L-arabinose) were found exclusively or almost exclusively in the cell water, demonstrating a rectilinear distribution pattern; the equilibrium distribution coefficients (q-values) obtained from the slopes of these rectilinear distribution curves are 0.256, 0.274, and 0.271 respectively. The fourth pentose, D-xylose, is apparently partially adsorbed. With the aid of the equation for solute distribution according to the association-induction (AI) hypothesis, the data for this sugar can also be fitted with a theoretical curve calculated on the basis of a q-value close to those for the other three pentoses. The close similarity of the q-values of pentoses which are sterically different, but have identical molecular weights, provides further confirmation of the "size rule", a prediction of the polarized multilayer (PM) theory of cell water, according to which, the q-values are as a rule primarily determined by the molecular weights of the solute and to a lesser extent dependent on its stereospecificity.     

Studies on the physical state of water in living cells and model systems. I. The quantitative relationship between the concentration of gelatin and certain oxygen-containing polymers and their influence upon the solubility of water for NA+ salts

The quantitative relationships between the concentrations of solutions of gelatin, polyvinylpyrrolidone, poly(ethylene oxide), polyvinylmethylether, and poly(ethylene glycol), and their ability to reduce the solubility of water for Na citrate are presented. The data in general are in harmony with the polarized multilayer theory of protein (and polymer) dominated water in vitro and in living cells.     

Studies on the physical state of water in living cells and model systems: IX. Theoretical significance of a straight line relationship between intracellular concentration of a partially excluded solute and its concentration in the bathing medium

The experimentally observed steady-level distribution of Na+ (25 degrees) and of D-glucose (0 degree c) in frog muscle were chosen as examples of solute distribution patterns observed in living cells, for comparison with those predicted by two theoretical models: one derived from the membrane-pump theory and the other from the association-induction (AI) hypothesis. Neither the distribution of Na+ nor that of D-glucose follows the pattern predicted by the membrane-pump models for solutes maintained at lower level than in the external medium, in which the plot of intracellular solute concentration as ordinate against different external concentrations as abscissa bends upward with increasing external solute concentration. Instead, both Na+ and D-glucose exhibit either straight line distribution with unchanging (below unity) slopes, or that of a hyperbola superimposed on such a straight line, both in agreement with the AI hypothesis.     

Relationship between Recrystallization Rate of Ice Crystals in Sugar Solutions and Water Mobility in Freeze-Concentrated Matrix

To better understand the relation between recrystallization rate and water mobility in freeze-concentrated matrix, isothermal ice recrystallization rates in several sugar aqueous solutions and self-diffusion coefficients of water component in corresponding freeze-concentrated matrix were measured. The sugars used were fructose, glucose, maltose, and sucrose. The sugar concentrations and temperature were varied so that ice contents for all samples were almost equal. Neither recrystallization rates nor diffusion coefficients depended uniformly on temperature. The recrystallization rates increased with increasing the diffusion coefficients, and a direct relationship was found between recrystallization rate and diffusion coefficient. This indicated that self-diffusion coefficient of water component in freeze-concentrated matrix is a useful parameter for predicting and controlling recrystallization rate in sugar solutions relevant to frozen desserts.     

Studies on Ion Accumulation in Muscle Cells

A comparison is made between the quantitative predictions of equilibrium ionic distribution in living cells according to the membrane theory (Donnan equilibrium) and according to the association-induction hypothesis. This comparison shows that both theories predict competitive effects of one permeant ion on the equilibrium concentration of another permeant ion; but within the limit of experimental accuracy only the association-induction model predicts quantitatively significant specific competition of one specified ion with the accumulation of another specified ion. The equilibrium distributions of K⁺, Rb⁺, and Cs⁺ ions in frog sartorius muscle were studied and quantitatively significant specific competition was demonstrated; these results favor the association-induction hypothesis (adsorption on cell proteins and protein complexes and partial exclusion from cell water). Based on this model we estimated that at 257deg;C, the apparent association constants for K⁺, Rb⁺, and Cs⁺ ion are 665, 756, and 488 (mole/liter)^-1. We found that the total concentration of adsorption sites (no less than 240 mmole/kg of fresh cells) agrees with the analytically determined concentrations of β- and γ-carboxyl groups of muscle cell proteins (260 to 288 mmole/kg).     

Characterization of Sugar and Polyphenolic Diversity in Floral Nectar of Different ‘Oblačinska’ Sour Cherry Clones

‘Obla?inska’ sour cherry, an autochthonous cultivar, is the most planted cultivar in Serbian orchards. Since fruit trees in temperate zone reward insects by producing nectar which ‘quality’ affects the efficiency of insect pollination, the aim of this study was analyzing of sugars and polyphenolics in floral nectar of 16 ‘Obla?inska’ sour cherry clones with different yielding potential. The contents of sugars and sugar alcohols were analyzed by ion chromatography, while polyphenolic profile was established using liquid chromatography/mass spectrometry technique. Fourteen sugars and six sugar alcohols were detected in nectar samples and the most abundant were fructose, glucose, and sucrose. Eleven polyphenols were quantified using available standards, while another 17 were identified according to their exact masses and characteristic fragmentations. Among quantified polyphenols, rutin, naringenin, and chrysin were the most abundant in nectar. Principal component analysis showed that some polyphenol components (naringin, naringenin, and rutin) together with sugars had high impact of spatial distribution of nectar samples on score plot.     

Studies on the physical state of water in living cells and model systems. VI. Concentration-dependent sustained volume changes of dialysis sacs containing aqueous solution of native and denatured protein, gelatin, and oxygen-containing polymers immersed in solutions of Na salt and of sugar and sugar alcohol

In this paper we studied the volume changes of dialysis sacs containing concentrated solutions of native and denatured proteins and of oxygen-containing polymers after immersion in aqueous solution of Na-citrate, D-glucose, and sorbitol of varying concentration. The results confirm the theory of cell volume regulation: volume changes of living cells in different solutions represent a balance between the tendency of intracellular proteins -- which exist in the fully extended conformation -- to polarize, sorb, and draw into the sac or cell more water and the opposite tendency to lose water from the sac or cell created by the lower level of the solutes in the cell or sac water than in the external medium. The lower level of the solutes is the consequence of the reduced solvency of the polarized water in the sac or cell water for large and complex solutes like sugar and free amino acids. This study adds another important physico-chemical attribute of the living cell that can be duplicated by aqueous solutions of gelatin, oxygen-containing polymers like PEO and PEG as well as urea-denatured proteins but not, or only weakly so, by aqueous solutions of native proteins or SDS-denatured proteins.     

Development of a buffer system for dialysis of bovine spermatozoa before freezing. II. Effect of sugars and sugar alcohols on posthaw motility

Two experiments were conducted to evaluate the effect of different levels of sugars (glucose, lactose and raffinose) and the effect of those sugars (C(3) to C(6)) or their correspondent sugar alcohols on the dialysis of bovine semen. First, the effect of isosmotic solutions of glucose, lactose or raffinose at five different levels (0, 25, 50, 75, 95% V/V) on sperm motility of semen dialyzed prior to freezing were studied. These levels were used in extenders and dialysates, and the final volume was complemented with Piperazine-N-N-BIS (2-ethane sulfonic acid (PIPES) titrated to pH 7.0 with TRIS (hydroxymethyl) amino-methane (TRIS) to form PIPEST or a 1:1 (V/V) combination between PIPEST and sodium citrate solutions. In the second experiment, 30% of the buffer volume contained solutions of sugars (C(3) or C(6)) or their correspondent sugar alcohol, and the final volume was completed with PIPEST-citrate buffer. Semen aliquots were extended (1:10) and dialyzed (1:50) for 2 h while cooling from 37 to 5 degrees C in semipermeable dialysis bags of 12,000 to 14,000 molecular weight cut off. The samples were frozen in pellets 1 h after dialysis was terminated. Sperm survival was significantly higher in PIPEST-citrate than in PIPEST buffer alone (P<0.05). No significant difference (P>0.05) was obtained between the use of glucose or lactose or between lactose and raffinose. High levels of sugar appeared to be detrimental to sperm motility of fresh and thawed semen samples. Motility of cells extended in buffers containing 30% (V/V) isosmotic solutions of glucose, galactose, ribose, xylose, arabinose or their correspondent sugar alcohols was significantly higher (P<0.05) than their motility in extenders without sugar.     

Fluorescence-polarization studies on binding of 4-methylumbelliferyl beta-D-galactopyranoside to Ricinus communis (castor-bean) agglutinin.

The binding of Ricinus communis (castor-bean) agglutinin 1 to saccharides was studied by equilibrium dialysis and fluorescence polarization by using the fluorescently labelled sugar 4-methylumbelliferyl beta-D-galactopyranoside. No appreciable change in ligand fluorescence of 4-methylumbelliferyl beta-D-galactopyranoside was considerably polarized on its binding to the lectin. The association constants obtained by Scatchard analysis of equilibrium-dialysis and fluorescence-polarization data do not differ much from each other, and at 25 degrees C, Ka = 2.4 (+/- 0.2) X 10(4)M-1. These values agree reasonably well with that reported in the literature for Ricinus agglutinin 1. The number of binding sites obtained by the different experimental procedures is 1.94 +/- 0.1 per molecule of 120 000 daltons and is equal to the reported value of 2. The consistency in the values of Ka and number of binding sites indicate the absence of additional subsites on Ricinus agglutinin 1 for its specific sugars. In addition, the excellent agreement between the binding parameters obtained by equilibrium dialysis and fluorescence polarization indicate the potential of ligand-fluorescence-polarization measurements in the investigation of lectin-sugar interactions.     

Freeze-thaw injury to isolated spinach protoplasts and its simulation at above freezing temperatures

Possibilities to account for the mechanism of freeze-thaw injury to isolated protoplasts of Spinacia oleracea L. cv. Winter Bloomsdale were investigated. A freeze-thaw cycle to −3.9 C resulted in 80% lysis of the protoplasts. At −3.9 C, protoplasts are exposed to the equivalent of a 2.1 osmolal solution. Isolated protoplasts behave as ideal osmometers in the range of concentrations tested (0.35 to 2.75 osmolal), arguing against a minimum critical volume as a mechanism of injury. Average protoplast volume after a freeze-thaw cycle was not greatly different than the volume before freezing, arguing against an irreversible influx of solutes while frozen. A wide variety of sugars and sugar alcohols, none of which was freely permeant, were capable of protecting against injury which occurred when protoplasts were frozen in salt solutions. The extent of injury was also dependent upon the type of monovalent ions present, with Li = Na > K = Rb = Cs and Cl ≥ Br > I, in order of decreasing protoplast survival. Osmotic conditions encountered during a freeze-thaw cycle were established at room temperature by exposing protoplasts to high salt concentrations and then diluting the osmoticum. Injury occurred only after dilution of the osmoticum and was correlated with the expansion of the plasma membrane. Injury observed in frozen-thawed protoplasts was correlated with the increase in surface area the plasma membrane should have undergone during thawing, supporting the contention that contraction of the plasma membrane during freezing and its expansion during thawing are two interacting lesions which cause protoplast lysis during a freezethaw cycle.     

Kinetics of 3-O-methyl-D-glucose transport in isolated rat hepatocytes.

3-O-Methyl-D-glucose transport across the plasma membrane of isolated rat hepatocytes was followed for net entry of the sugar into sugar-free cells (zero trans entry), net exit of sugar into sugar-free medium (zero trans exit) and for unidirectional entry and exit fluxes when cells had been equilibrated with sugar in the extracellular medium (equilibrium exchange entry and exit). These measurements were performed at 20 degrees C and pH 7.4 by the use of simple manual methods. Initial rates of transport showed a Michaelis--Menten dependency on the sugar concentration at the cis side of the membrane over the range of concentrations tested (100 microM to 100 mM). Transport was found to be symmetrical with no evidence of substrate stimulation of transport from the trans side of the membrane. Parameters (mean values +/- S.E.M.) of transport were estimated as Vmax. 86.2 +/- 9.7 mmol/litre of cell water per min and Km 18.1 +/- 5.9 mM for exchange entry, Vmax. 78.8 +/- 5.3 mmol/litre of cell water per min and Km 17.6 +/- 3.5 mM for exchange exit, Vmax. 84.1 +/- 8.4 mmol/litre of cell water per min and Km 16.8 +/- 4.6 mM for zero trans exit.     

植物组织中糖与糖醇乙酰化及毛细管气相色谱分析

介绍了一种用1-甲基咪唑为溶剂和催化剂、盐酸羟胺和乙酸酐为肟化和乙酰化试剂,对植物样品中糖与糖醇进行乙酰化衍生化后的气相色谱分离和质谱鉴定的分析方法.并对糖与糖醇乙酰化影响较大的反应温度、反应时间、反应物组成和反应物浓度等条件进行了比较研究,确定了糖与糖醇乙酰化各步反应的最佳反应温度和反应时间,分析了各组分间相互作用及其用量对衍生化效率的影响.对多种糖与糖醇乙酰化产物进行毛细管气相色谱分离、FID检测及GC-MS结构鉴定.研究证明,在合适条件下应用此方法对糖与糖醇进行乙酰化,反应完全,产物单一,能得到理想的分离、检测和定量分析效果.适用于微量植物组织中多种单糖、双糖及其糖醇的定量分析.     

Distinct Cellular and Organismic Responses to Salt Stress

We have compared metabolic effects of high salinity betweenplants and cell suspension cultures from the facultative halophyteMesembryanthemum crystallinum (common ice plant). This plantshows developmentally-programmed inducibility for a switch fromC₃-photosynthesis to CAM (Crassulacean Acid Metabolism). Themetabolic switch is enhanced by environmental factors such asdrought, low temperature, and, most effectively, soil salinity.CAM induction is dependent on organized leaf tissue and cannotbe elicited by salt stress in suspension culture cells. In contrast,the accumulation of proline [Thomas et al. (1992) Plant Physiol.98: 626] is induced by NaCl in cultured cells as well as inplants and must be considered a cellular response to stress.We have extended our observations to include another trait ofsalt- and low-temperature-stress responses in the ice plant,the accumulation of putative osmoprotective sugars and sugaralcohols. In whole plants the cyclic sugar alcohol, pinitol,accumulates to amounts that approach 1 M during stress, whilein suspension cells no increase in sugar alcohols is observed.The distribution of carbon to different sugars is markedly differentbetween cells and plants under stress. Particularly obviousis the distinction between cell types in the different compositionof sugars and polyols, as exemplified by the epidermal bladdercells of ice plants. Ion contents and the content of sugarsand sugar alcohols of bladder cells indicate that Na⁺, Cl^–,pinitol and an unknown carbohydrate compound provide osmoticpressure in these cells, while organic anion concentrationsare low. With the ice plant, we conclude that cells in culturemimic only partly the stress response mechanisms of intact plantsand we hypothesize that communication between different tissuesis required to mount a complete environmental stress response. ⁴ Present address: Department of Botany Oklahoma State University,Stillwater, OK, 74078, U.S.A.     

Variable H+/substrate stoicheiometries in Rhodotorula gracilis are caused by a pH-dependent protonation of the carrier(s).

Two carrier-mediated systems transport sugars in the yeast Rhodotorula gracilis depending on the pH. One system, with higher affinity for sugars, catalyses a symport of protons with sugar, whereas the other system, having lower affinity, is independent of protons. This was shown in three different ways. (1) At low pH, where only the high-affinity system works, a H+/sugar stoicheiometry of 1 was found. An increase of the pH and of the sugar concentration, which allowed the low-affinity system to operate, brought about a drop of the stoicheiometry to values below 1. (2) During H+ symport the influx of positive charge was electrically compensated by an equivalent efflux of K+ from the cells. At high pH and high sugar concentrations this stoicheiometry of K+ and sugar decreased concomitant with the H+/sugar stoicheiometry. (3) At pH 7.5 both transport systems were operating, as shown by biphasic saturation kinetics. Under these conditions only the high-affinity transport was found to be electrogenic. These results agree with the theory of an electrogenic H+/sugar symport where changes in the affinity for substrate are brought about by reversible protonation and deprotonation of the carrier.     

罗汉果果肉中糖类物质组成与含量分析

罗汉果果实中富含糖分,糖类物质的组成及其含量对果实的内在品质有重要影响,然而多年来对其品质的研究多集中在罗汉果苷上,果实中可溶性糖种类与含量迄今尚未见有系统地报道。该研究以干燥的罗汉果果实为材料,采用PMP柱前衍生化一高效液相色谱紫外检测法、高效液相色谱示差折光检测法分别检测果肉中可溶性糖的种类与含量,并进行方法学考察。结果表明:PMP柱前衍生化一高效液相色谱紫外检测法只能检出罗汉果果实中存在的2种还原性醛糖——葡萄糖、甘露糖;而高效液相色谱示差折光检测法则可一次性检出葡萄糖、果糖、蔗糖、棉籽糖、多糖5种糖分。与柱前衍生化法相比,高效液相色谱示差折光检测法更适合用来全面分析罗汉果果实中糖分的种类和含量。不同罗汉果品种果实中糖的组分一致,但含量有显著差别。另外,样品的干燥方式会影响果实中的总糖及各组分的相对含量。冻干果肉中蔗糖和葡萄糖相对含量最高,烘干则导致蔗糖和葡萄糖下降,果糖与多糖相对含量增加。     

Turgor regulation of sucrose transport in sugar beet taproot tissue

Sink tissues that store osmotically active compounds must osmoregulate to prevent excessively high turgor. The ability to regulate turgor may be related to membrane transport of solutes and thus sink strength. To study this possibility, the kinetics of sugar uptake were determined in sugar beet (Beta vulgaris L.) taproot tissue discs over a range of cell turgors. Sucrose uptake followed biphasic kinetics with a high affinity saturating component below 20 millimolar and a low affinity linear component at higher concentrations. Glucose uptake exhibited only simple saturation type kinetics. The high affinity saturating component of sucrose and glucose uptake was inhibited by increasing cell turgor (decreasing external mannitol concentrations). The inhibition was evident as a decrease in V_max but no effect on K_m. Sucrose uptake by tissue equilibrated in dilute buffer exhibited no saturating component. Ethylene glycol, a permeant osmoticum, had no effect on uptake kinetics, suggesting that the effect was due to changes in cell turgor and not due to decreased water potential per se. p-(Chloromercuri)benzene sulfonic acid (PCMBS) inhibited sucrose uptake at low but not high cell turgor. High cell turgor caused the tissue to become generally leaky to potassium, sucrose, amino acids, and reducing sugars. PCMBS had no effect on sucrose leakage, an indication that the turgor-induced leakage of sucrose was not via back flow through the carrier. The ability of the tissue to acidify the external media was turgor dependent with an optimum at 300 kilopascals. Acidification was sharply reduced at cell turgors above or below the optimum. The results suggest that the secondary transport of sucrose is reduced at high turgor as a result of inhibition of the plasma membrane ATPase. This inhibition of ATPase activity would explain the reduced V_max and leakiness to low molecular weight solutes. Cell turgor is an important regulator of sucrose uptake in this tissue and thus may be an important determinant of sink strength in tissues that store sucrose.     

History of the membrane (pump) theory of the living cell from its beginning in mid-19th century to its disproof 45 years ago--though still taught worldwide today as established truth

The concept that the basic unit of all life, the cell, is a membrane-enclosed soup of (free) water, (free) K+ (and native) proteins is called the membrane theory. A careful examination of past records shows that this theory has no author in the true sense of the word. Rather, it grew mostly out of some mistaken ideas made by Theodor Schwann in his Cell Theory. (This is not to deny that there is a membrane theory with an authentic author but this authored membrane theory came later and is much more narrowly focussed and accordingly can at best be regarded as an offshoot of the broader and older membrane theory without an author.) However, there is no ambiguity on the demise of the membrane theory, which occurred more than 60 years ago, when a flood of converging evidence showed that the asymmetrical distribution of K+ and Na+ observed in virtually all living cells is not the result of the presence of a membrane barrier that permits some solutes like water and K+ to move in and out of the cell, while barring--absolutely and permanently--the passage of other solutes like Na+. To keep the membrane theory afloat, submicroscopic pumps were installed across the cell membrane to maintain, for example, the level of Na+ in the cell low and the level of K+ high by the ceaseless pumping activities at the expense of metabolic energy. Forty-five year ago this version of the membrane theory was also experimentally disproved. In spite of all these overwhelming evidence against the membrane-pump theory, it still is being taught as verified truth in all high-school and biology textbooks known to us today. Meanwhile, almost unnoticed, a new unifying theory of the living cell, called the association-induction hypothesis came into being some 40 years ago. Also little noticed was the fact that it has received extensive confirmation worldwide and has shown an ability to provide self-consistent interpretations of most if not all known experimental observations that are contradicting the membrane-pump theory as well as other observations that seem to support the membrane pump theory.