PERIODIC CHANGE IN THE TENSION AT THE SURFACE OF ACTIVATED NON-NUCLEATE FRAGMENTS OF SEA-URCHIN EGGS

Unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus , were separated into two fragments by centrifugal force. The enucleate fragment (merogone) was subsequently activated by treating it with butyric acid and the tension at the surface was continuously measured by a compression method. The activated merogone was found to exhibit cyclic changes in tension, with a temporal pattern very similar to that of the changes accompanying the division cycle of normally fertilized eggs. This indicates quantitatively the presence in the cytoplasm of some periodic activity which can be triggered without nuclear control. Further, a periodic thickening of the intrahyaloplasmic space of the activated merogone, as noted by K ojima (14), was confirmed on the basis of extended observation.     

THE DEFORMABILITY AND THE WETTING PROPERTIES OF LEUCOCYTES AND ERYTHROCYTES

The resistance to deformation of polymorphonuclear neutrophile leucocytes under the conditions of our observations has been shown to be on the average considerably less than the resistance to deformation of large mononuclear leucocytes. It is recognized of course that the viscosity of leucocytes, as of other cells, may be markedly influenced by osmotic conditions (17), by the reaction of the suspending medium (18, 19), by temperature, or by injury (20, 21). Although the conditions of our observations were quite different from those of the body, they were nevertheless closely similar to those of simultaneous phagocytosis experiments in which the cells functioned exceedingly well (3). Moreover E. R. and E. L. Clark (22) have noted that polymorphonuclear leucocytes in the tails of living tadpoles were more fluid than the macrophages. And Goss (23) in microdissecting human polymorphonuclear neutrophiles reports that they are more fluid than the clasmatocytes and monocytes studied by Chambers and Borquist (24). Other types of leucocytes have in our experience seemed to fall between the large mononuclear and the polymorphonuclear leucocytes in their average resistance to the interfacial tensions. The leucocyte of each type studied is surrounded by an exceedingly delicate membrane. This membrane appears under the dark-field microscope as a pale, silvery line not distinguishable by inspection alone from a simple phase boundary between two immiscible liquids. That this is a membrane, however, and not a mere interface between immiscible phases, seems certain. In the first place the cell cytoplasm and the suspending medium are not immiscible. When the cell organization is broken down by the interfacial tension the greater part of the cell contents is immediately dissolved or dispersed. Goss (23) has noted that when the membrane is torn with a microdissection needle disintegration at once spreads over the membrane and the cytoplasm undergoes profound change. Moreover it is improbable that a simple phase boundary could exist in the presence of so much protein, lipoid, and other surface active materials as are present in protoplasm; the tendency of these substances to lower the free interfacial energy must necessarily tend to their adsorption in the interface until, if sufficient material is available at the interface, an adsorption film or membrane may be formed. Kite (25), in a pioneer microdissection study, described the polymorphonuclear leucocyte as "naked" protoplasm. The contradiction between this statement and those just made is more apparent than real. For the capacity swiftly to form a limiting membrane between itself and other liquids is an attribute of "naked" protoplasm, as has been shown by the beautiful experiments of Chambers (20). The present study of the wetting properties of leucocytes shows that their external membranes are hydrophilic, a character suggesting a surface in which proteins, probably bound water and salts (27), possibly the polar radicles of soaps or fatty acids, rather than non-polar lipoid groupings, are predominantly exposed. This makes it the more remarkable that a cell of such fluidity as for instance the polymorphonuclear leucocyte, composed largely of water and of water-soluble materials, should maintain its integrity in an aqueous medium with the aid of a membrane so delicate and so mobile. The mobility of the membrane, frequently extended in forming new pseudopodia or spreading over the surface of particles being ingested, must require constant entrance into and exit from the membrane of component materials, and their constant reorganization there. The limiting factors in the reformation of such a membrane would be the amounts of adsorbable materials available and their rates of movement up to the surface rather than the time required for orientation there, since the latter phenomenon is exceedingly rapid. Harkins (29), for instance has calculated that at a water-water vapor interface at 20°C., from the area occupied by one molecule of water, a molecule would jump out into the vapor and a vapor molecule would fall into this area of the surface 7,000,000 times in one second; the time of orientation of the water molecule he estimates to be of the order of 1/100,000,000 second or less. The mammalian erythrocyte possesses a surface membrane capable of being folded and of withstanding tension in the interface. This has also been stretched by microdissection needles (21). The surface of the erythrocyte, as evidenced by its wetting properties, is relatively hydrophobic, relatively non-polar in character, as compared with the leucocyte. Evidence indicating that the erythrocyte surface contains both lipoid and protein components has been summarized in earlier papers (8, 30). We have little to add here other than to point out that the wetting properties of the chicken erythrocyte surface are similar to those fully described for the mammal. A serious source of error in certain isoelectric point determinations is discussed.     

PERIODIC CHANGES IN THE CONTENT OF PROTEIN-BOUND SULFHYDRYL GROUPS AND TENSION AT THE SURFACE OF STARFISH OOCYTES IN CORRELATION WITH THE MEIOTIC DIVISION CYCLE*

The sulfhydryl content of protein and the tension at the surface were measured for starfish oocytes from the first meiotic division to the cleavage stage. A cyclic change in both the protein-SH and the tension at the surface was found to accompany the division cycle, including the first and second meiotic divisions. It is concluded that the unequal meiotic divisions share the same character with the equal divisions of cleavage, with respect to changes both in the protein-SH and the tension at the surface.     

Studies of excitable membrane formed on the surface of protoplasmic drops isolated from Nitella II. Tension at the surface of protoplasmic drops

A protoplasmic drop isolated from an internodal cell of Nitella became electrically excitable in a solution containing 0.5 mM NaCl, 0.5 mM KNO₃, 1mM Ca(NO₃)₂ and 2mM Mg(NO₃)₂. A thermodynamic property of the excitable membrane was characterized in terms of tension at the surface of the protoplasmic drop. This was determined by the compression method and/or by the sessile-drop method. The surface tension of the membrane was obtained as a function of the composition of the salts in the external solution, and the time during the formative period of the excitable surface membrane. The results are summarized as follows:

1. 1. The surface of the protoplasmic drop increased with time starting from 0.003 dyne/cm and approached a steady value of about 0.1 dyne/cm within 1 h after the drop was placed in the test solution described above. The membrane became electrically excitable when the surface tension attained the steady value.

2. 2. Increase of concentration of either Na⁺ or K⁺ in the solution induced a sudden decrease of the surface tension, which followed a suppression of the excitability. The critical concentration of Na⁺ or K⁺ was about 10 mM.

3. 3. The surface tension remained constant at about 0.1 dyne/cm in a Ca²⁺ concentration ranging between about 0.1 and 10 mM. At this concentration the drop was excitable. Below and above this range of Ca²⁺ concentration, the surface tension changed sharply with concentration, and the excitability disappeared. At about 0.1 mM Ca²⁺ concentration a discrete variation of the surface tension was observed.

4. 4. The surface tension of the drop stayed constant at 0.1 dyne/cm in the range between 1 and 10 mM of Mg²⁺ concentration. Above and below this range of Mg²⁺ concentration, the surface tension increased sharply with the variation of Mg²⁺ concentration.

These results indicate that the protoplasmic drop retains its excitability in a limited range of salt composition in the external solution. This implies that the excitable membrane of the drop must be very labile in its structure against external perturbations such as electrical stimulus and/or slight variation of salt composition in the solution.     

THE SURFACE TENSION OF PHYSIOLOGICAL SOLUTIONS : DIFFICULTIES OF MEASUREMENT AND INTERPRETATION.

1. Data are given to show that surface tension values obtained by the ring method cannot be considered reliable when absolute values are desired. 2. Data are given to show that, in the case of solutions of semicolloids, surface tension values obtained for a definite period of time following the formation of a new interface cannot always be consistently reproduced by the ring method. 3. It is shown that in the case of solutions of semicolloids, equilibrium values are not readily obtained and should generally not be assumed to have been reached after any definite period of time. 4. The general difficulty of interpreting surface tension values obtained in the case of solutions of semicolloids, by any method, is emphasized.     

THE MECHANISM OF THE WATER EXPULSION VESICLE OF THE CILIATE TETRAHYMENA PYRIFORMIS

Analysis of high-speed (150 frames/sec) cinematographs of the filling and expulsion of the water expulsion vesicle of Tetrahymena pyriformis shows that the vesicle fills as water is pumped into it by contractions of at least four ampullary sacs which are continuous with the endoplasmic reticulum. When filled, the vesicle is pressed against its two excretory pores by cyclotic movements of the cytoplasm. This pressure closes the apertures of the ampullae, preventing backflow from the vesicle into them, and also spreads the pellicle of and at the pore, thereby stretching and rupturing the pore-sealing membrane. The vesicle is then invaginated by the cytoplasmic pressure, driving fluid out of the pore. The pore-sealing membrane then reforms, apparently by constriction, and the vesicle is again filled. Electron micrographs show that crisscrossed pore-microtubules extend from the pore to the openings of the ampullae, anchoring the vesicle in place. Each pore is surrounded by a stack of at least 11 ring-microtubules, to which the anchoring pore-microtubules are attached. The pore-microtubules appear to exert tension which assists in spreading the pore, aiding cyclotic pressures in rupturing the pore-sealing membrane. A possible mechanism for the cyclotic pressure and ampullary contraction is proposed.     

嗜菌紫膜表面电位和质子泵效率间的关系

本文研究了中性红与紫膜结合,并用直接滴定法和测定结合量法,求得了结合于膜上之中性红本征PK和在不同离子强度时的表观PKa值.从它们的差值中计算得到紫膜表面电位.并得不同盐浓度的表面电位和质子泵效率作了比较.结果说明.阳离子对质子泵效率的影响不能完全用表面电位的变化来说明.这一结果暗示了一定的阳离子对于紫膜质子泵功能的完成有着特殊作用.     

Effect of surfactant concentration, compression ratio and compression rate on the surface activity and dynamic properties of a lung surfactant

This paper reports dynamic surface tension experiments of a lung surfactant preparation, BLES, for a wide range of concentrations, compression ratios and compression rates. These experiments were performed using Axisymmetric Drop Shape Analysis-Constrained Sessile Drop (ADSA-CSD). The main purpose of the paper is to interpret the results in terms of physical parameters using the recently developed Compression-Relaxation Model (CRM). In the past, only the minimum surface tension was used generally for the characterization of lung surfactant films; however, this minimum value is not a physical parameter and depends on the compression protocol. CRM is based on the assumption that the dynamic surface tension response is governed by surface elasticities, adsorption and desorption of components of the lung surfactant. The ability of CRM to fit the surface tension response closely for a wide variety of parameters (compression ratio, compression rate and surfactant concentration) and produce sensible values for the elastic and kinetic parameters supports the validity of CRM.     

Influence of ion occupancy and membrane deformation on gramicidin A channel stability in lipid membranes.

The average lifetime of gramicidin A channels in monoolein/decane bilayer membranes was measured. The results support the hypothesis of channel stabilization by ion occupancy. The effects of electric field and salt concentration are consistent with the expected effects on both occupancy and membrane compression. The lifetime in asymmetric solutions with divalent cation blockers on one side of the membrane shows a voltage dependence such that the lifetime decreases for positive voltages applied from the blocking side and increases for negative voltages. This result strongly supports the occupancy hypothesis. The lifetime increases with permeant ion concentration, and at the one molar level it also increases with voltage. The voltage dependence of lifetime for a low concentration of permeant ion depends on the total salt level. The results for these conditions are consistent with the assumption that membrane compression also influences the lifetime, even for the "soft" solvent-containing membrane considered here. It is proposed that the channel nearest neighbor lipids need not be fixed in a plane at the channel end. Using a liquid crystal model it may then be shown that surface tension is the major component of the membrane deformation free energy, which may explain the significant effects of the membrane compression on the lifetime.     

TENSION AT THE HIGHLY STRETCHED SURFACE OF SEA-URCHIN EGGS*

Unfertilized eggs of the sea urchin, Paracentrotus lividus, were placed between two parallel plates and flattened by a definite force to 20% of their original diameter, with two-fold increase in their surface area. The resulting tension at their surface was calculated from the relation of force and deformation. In spite of this extensive stretching, the tension was found to be not more than 0.2 dyne/cm, while under conditions involving mild stretching (3%) the tension still amounted to 0.12 dyne/cm. These results do not support Mela's theory (7, 8), which predicts a transition of the mechanical properties of the egg surface from a ‘subelastic’ to ‘elastic’ state when the surface is stretched to beyond 34% of its initial area.     

Thermoelasticity of large lecithin bilayer vesicles.

Micromechanical experiments on large lecithin bilayer vesicles as a function of temperature have demonstrated an essential feature of bilayer vesicles as closed systems: the bilayer can exist in a tension-free state (within the limits of experimental resolution, i.e., less than 10(-2) dyn/cm). Furthermore, because of the fixed internal volume, there is a critical temperature at which the vesicle becomes a tension-free sphere. Below this temperature, thermoelastic tension builds up in the membrane and the vesicle's internal pressure increases while the surface area remains constant. Above this temperature, the vesicle's surface area increases while the tension and internal pressure are negligible. Without mechanical support, the vesicles fragment into small vesicles because they have insufficient surface rigidity. In the upper temperature range we have measured the increase of surface area with temperature. These data established the thermal area expansivity to be 2.4 X 10(-3)/degrees C. At constant temperature, we used either pipet aspiration with suction pressures up to 10(4) dyn/cm2 or compression against a flat surface with forces up to 10(-2) dyn to produce area dilation of the vesicle surface on the order of 1%. The rate of increase of membrane tension with area dilation was calculated, which established the elastic area compressibility modulus to be 140 dyn/cm. The tension limit that produced lysis was observed to be 3-4 dyn/cm (equivalent to 2-3% area increase). The product of the elastic area compressibility modulus, the thermal area expansivity, and the temperature gives the reversible heat of expansion at constant temperature for the bilayer. This value is 100 ergs/cm2 at 25 degrees C, or approximately 5 kcal/mol of lecithin. Similarly, the product of the thermal area expansivity multiplied by the area compressibility modulus determines the rate of increase of thermoelastic tension with decrease in temperature when the area is held constant, i.e., -0.34 dyn/cm/degrees C.     

Interactions between plasma proteins and pulmonary surfactant: surface balance studies

The influence of human fibrinogen, alpha-globulin, and albumin on the properties of monolayers of pulmonary surfactant under dynamic compression and expansion has been studied at 37 degrees C. Each of the proteins altered some of the properties of the normal compression and expansion isotherms of surfactant such that characteristics deemed desirable for proper lung function were impaired. The order of potency of these effects was fibrinogen greater than globulin greater than albumin. The proteins (a) decreased the maximum surface pressure (equivalent to the minimum surface tension) which the surfactant monolayers attained on compression, (b) decreased the areas occupied per mole of lipid phosphorus when the monolayers were at surface tensions of 20 and 12 mN.m-1, (c) reduced the areas of the hysteresis between compression and expansion isotherms, and (d) decreased the rate of change of surface tension with area at the point of initial expansion of the monolayers. The proteins might compete with surfactant lipid for available space at the interface, especially at low film compression. They might also enhance the desorption of lipid from the monolayer. The findings are consistent with the loss of pulmonary function and presence of edema that occur in adult respiratory distress syndrome being contributed to by plasma proteins interfering with surfactant function.     

Increase in tension at the surface of protoplast as a sign of cell wall formation inBoergesenia forbessi

Protoplasts prepared from thalli ofBoergesenia forbesii were subjected to the measurement of tension at the surface by means of the suction method. The tension at the surface just after completion of spheration was 0.2–0.4 dyne/cm irrespective of the temperature. Since this value is of the same order of magnitude as those measured in other species of cells without a cell coat, it is suggested that the protoplast just after spheration is covered with the plasma membrane. The measured tension at the surface was constant and not affected by the degree of deformation of the protoplast, suggesting that the surface of the protoplast is not elastic. After some time the tension began to increase abruptly. Both the latent time elapsed prior to the increase in the tension and the rate of tension increase were strongly dependent on the temperature. As long as protoplasts were treated with cellulase, increase in the tension was completely inhibited, but it occurred soon after washing out of the cellulase. Protoplasts were stained with Calcoflour White at around the time when the tension began to increase. These results suggest that the cell wall formation begins at the time of abrupt increase in the tension at the surface.     

FACTORS CONTROLLING THE REASSEMBLY OF THE MICROVILLOUS BORDER OF THE SMALL INTESTINE OF THE SALAMANDER

Hydrostatic pressure, when applied to segments of the small intestine of the salamander, causes a tremendous reduction in number of microvilli and a loss of the terminal web. The intestinal epithelium strips off from its deeper layers at the level of the basement membrane. When the pressure is released and this epithelial sheet is allowed to recover, the microvilli and its terminal web reappear. Stages in the reformation of microvilli are described. In the earliest stages, foci of dense material seem to associate with the cytoplasmic surface of the apical plasma membrane. From this material, filaments appear and their regrowth is correlated with the extension of the microvilli. We suggest that the dense material nucleates the assembly of the filaments which, in turn, appear instrumental in the redevelopment of microvilli. This concept is supported by the existing literature. Further, since neither the microvilli nor the terminal web reappear on any surface but the apical surface, even though the apical and basal surfaces are bathed with the same medium, we suggest that information in the membrane itself or directly associated with the membrane dictates the distribution of the dense material which leads to the formation of the microvilli and ultimately to the polarity of the cell.     

Elastic area compressibility modulus of red cell membrane.

Micropipette measurements of isotropic tension vs. area expansion in pre-swollen single human red cells gave a value of 288 +/- 50 SD dyn/cm for the elastic, area compressibility modulus of the total membrane at 25 degrees C. This elastic constant, characterizing the resistance to area expansion or compression, is about 4 X 10(4) times greater than the elastic modulus for shear rigidity; therefore, in situations where deformation of the membrane does not require large isotropic tensions (e.g., in passage through normal capillaries), the membrane can be treated by a simple constitutive relation for a two-dimensionally, incompressible material (i.e. fixed area). The tension was found to be linear and reversible for the range of area changes observed (within the experimental system resolution of 10%). The maximum fractional area expansion required to produce lysis was uniformly distributed between 2 and 4% with 3% average and 0.7% SD. By heating the cells to 50 degrees C, it appears that the structural matrix (responsible for the shear rigidity and most of the strength in isotropic tension) is disrupted and primarily the lipid bilayer resists lysis. Therefore, the relative contributions of the structural matrix and lipid bilayer to the elastic, area compressibility could be estimated. The maximum isotropic tension at 25 degrees C is 10-12 dyn/cm and at 50 degrees C is between 3 and 4 dyn/cm. From this data, the respective compressibilities are estimated at 193 dyn/cm and 95 dyn/cm for structural network and bilayer. The latter value correlates well with data on in vitro, monolayer surface pressure versus area curves at oil-water interfaces.     

Bending Resistance and Chemically Induced Moments in Membrane Bilayers

Pure bending of a membrane bilayer is developed including different properties for each membrane half. Both connected and unconnected bilayer surfaces are treated. The bilayer bending resistance is the resultant of parallel surface compression “resistances.” The neutral surface is a function of the upper and lower surface compressibility moduli and does not necessarily coincide with the mid-surface. Alterations in the interfacial chemical free energy density (surface tension) on either face can create induced bending moments and produce curvature; even small changes can have a pronounced curvature effect. Chemically induced moments are considered as a possible mechanism for crenation of red blood cells.     

Adhesive strength of single muscle cells to basement membrane at myotendinous junctions

Whole muscles loaded to failure frequently fail at or near myotendinous junctions. The present investigation was directed toward determining the breaking stress and failure site of intact and injured myotendinous junction preparations consisting of muscle cells dissected free from surrounding parallel structures but still attached to tendon collagen fibers. These tests show that the breaking stress for intact myotendinous units is 2.7 x 10(5) N/m2, expressed relative to cell cross-sectional area. Failure occurs immediately external to the junction membrane between the cell membrane and lamina densa of the basement membrane. Site and stress at failure are independent of strain and strain rate over a biologically relevant range. Breaking stress in the plane of the membrane, corrected for membrane folding, is 1.2 X 10(4) N/m2. This value is not significantly greater than stress at maximum isometric tension for these cells at these sarcomere lengths. After compression injury, cells fail within the compression site at significantly lower stress (1.9 X 10(5) N/m2). These findings suggest that, in muscle strain injuries that occur under conditions simulated here, failure occurs at myotendinous junctions unless the muscle has suffered previous compression injury leading to failure within the muscle.     

Effects of hemoglobin and cell membrane lipids on pulmonary surfactant activity

These experiments characterize the effects of hemoglobin and erythrocyte membrane lipids on the dynamic surface activity and adsorption facility of whole lung surfactant (LS) and a calf lung surfactant extract (CLSE) used clinically in surfactant replacement therapy for the neonatal respiratory distress syndrome (RDS). The results show that, at concentrations from 25 to 200 mg/ml, hemoglobin (Hb) increased the minimum dynamic surface tension of LS or CLSE mixtures (0.5 and 1.0 mumol/ml) from less than 1 to 25 dyn/cm on an oscillating bubble apparatus at 37 degrees C. Similarly, erythrocyte membrane lipids (0.5-3 mumol/ml) also prevented LS and CLSE suspensions (0.5-2.0 mumol/ml) from lowering surface tension below 19 dyn/cm under dynamic compression on the bubble. Surface pressure-time adsorption isotherms for LS suspensions (0.084 and 0.168 mumol phospholipid/ml) were also adversely affected by Hb (0.3-2.5 mg/ml), having a slower adsorption rate and magnitude. Significantly, these inhibitory effects of Hb and membrane lipids could be abolished if LS and CLSE concentrations were raised to high levels. In complementary physiological experiments, instillation of Hb, membrane lipids, or albumin into excised rat lungs was shown to cause a decrease in pressure-volume compliance. This decreased compliance was most prominent in lungs made partially surfactant deficient before inhibitor delivery and could be reversed by supplementation with active exogenous surfactant. Taken together, these data show that molecular components in hemorrhagic pulmonary edema can biophysically inactivate endogenous LS and adversely affect lung mechanics. Moreover, exogenous surfactant replacement can reverse this process even in the continued presence of inhibitor molecules and thus has potential utility in therapy for adult as well as neonatal RDS.     

Comparison of inhibitory effects of oxygen radicals and calf serum protein on surfactant activity

The effects of the reactive oxygen species (ROS) superoxide anion (O2*-) and hydroxyl radical (*OH) on the surface tension lowering properties of bovine lipid extract surfactant (BLES) were compared to the effects of calf serum protein (CSP) in a captive bubble surfactometer (CBS). O2*- was generated from xanthine/xanthine oxidase (X/XO), and *OH was generated by the Fenton reaction. ROS were demonstrated by electron spin resonance (ESR) using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin trap. Lipid peroxidation was measured using the thiobarbituric acid method. *OH had broad inhibitory effects on surface tension parameters, including adsorption, minimum surface tension, percentage film area change and film compressibility. O2*- showed inhibitory effects on adsorption, film area change and film compressibility but had no significant effect on minimum surface tension. Both O2*- and *OH treatment were associated with a large 'squeezeout' plateau around 20-25 mN/m in the surface tension-area relation, indicating poor film organization during the compression phase. At the concentrations used, ROS were associated with lipid peroxidation of BLES, which also demonstrated radical scavenging properties. Calf serum protein produced inhibitory effects on adsorption, minimum surface tension and percentage film area change that were quantitatively similar to those produced by *OH. The effects on film compression were significantly greater and qualitatively different from those seen with either O2*- or *OH. We conclude that the inhibition of BLES surface activity by ROS and inhibitory proteins can be distinguished in the captive bubble surfactometer and, particularly, by changes in the film compressibility modulus.