STUDIES ON PERMEABILITY OF MEMBRANES : VI. MENSURATION OF THE DRIED COLLODION MEMBRANE (CALCULATION OF DIMENSIONS AND OF RELATIONS TO CERTAIN BIOLOGICAL MEMBRANES).

The flat type of dried collodion membrane used by Michaelis and his associates in numerous investigations has been subjected to mensuration in order that the dimensions of these membranes may be placed on record. The membranes had a functioning area of about 30 cm., were approximately 0.1 mm. in thickness and were composed on the average of 87 per cent by volume of collodion and 13 per cent by volume of pores. In reviewing some of the previously reported results of diffusion experiments with non-electrolytes in the light of the calculated values for the total pore area for the same membranes additional evidence was presented to show that a smaller molecule (acetone) probably utilizes a much larger percentage of the total pore area for its diffusion than is available for a larger molecule (glycerol). By using the figures of Fricke and McClendon for the thickness of the membrane of the red blood cell some comparisons were drawn between the dried collodion membrane as a model for certain biological membranes and the red blood cell membrane. In these comparisons emphasis was placed on the exaggerated importance of small electromotive forces and very slight permeabilities when these were associated with membranes of such extreme thinness as the red blood cell membrane.     

THE STRUCTURE OF THE COLLODION MEMBRANE AND ITS ELECTRICAL BEHAVIOR : IX. WATER UPTAKE AND SWELLING OF COLLODION MEMBRANES IN AQUEOUS SOLUTIONS OF ORGANIC ELECTROLYTES AND NON-ELECTROLYTES

1. Dried collodion membranes are known to swell in water and to the same limited extent also in solutions of strong inorganic electrolytes (Carr and Sollner). The present investigation shows that in solutions of organic electrolytes and non-electrolytes, the swelling of dried collodion membranes is not as uniform, but depends on the nature of the solute. 2. The solutions of typically "hydrophilic" substances, e.g., glycerine, glucose, and citric acid, swell collodion membranes only to the same extent as water and solutions of strong electrolytes. In solutions of typically carbophilic substances (e.g., butyric acid, valeric acid, isobutyl alcohol, valeramide, phenol, and m-nitrophenol) the swelling of the membranes is much stronger than in water, according to the concentration used. For the brand of collodion used the swelling in 0.5 M solution was in some cases as high as 26 per cent of the original volume, as compared to 6 to 7 per cent in water. Therefore, in these solutions the "water-wetted dried" collodion membrane is not rigid, inert, and non-swelling, but behaves as a swelling membrane. 3. The solutes which cause an increased swelling of the membranes are accumulated in the latter, the degree of accumulation being markedly parallel with the degree of their specific swelling action. 4. The anomalously high permeabilities of certain carbophilic organic solutes reported by Michaelis, Collander, and Höber find an explanation in the specific interaction of these substances with collodion. 5. The use of the collodion membrane as a model of the ideal porous membrane is restricted to those instances in which no specific interaction occurs between the solute and the collodion.     

THE EFFECT OF SALT CONCENTRATION OF THE MEDIUM ON THE RATE OF OSMOSIS OF WATER THROUGH THE MEMBRANE OF LIVING CELLS

1. Using the unfertilized egg of the sea urchin, Arbacia, as osmometer, it was found that the rate with which water enters or leaves the cell depends on the osmotic pressure of the medium: the velocity constant of the diffusion process is higher when the cell is in concentrated sea water, and lower when the sea water medium is diluted with distilled water. Differences of more than tenfold in the value of the velocity constant were obtained in this way. When velocity constants are plotted against concentration of medium, a sigmoid curve is obtained. 2. These results are believed to indicate that cells are more permeable to water when the osmotic pressure of the medium is high than when it is low. This relation would be accounted for if water should diffuse through pores in a partially hydrated gel, constituting the cell membrane. In a medium of high osmotic pressure, the gel is conceived to give up water, to shrink, and therefore to allow widening of its pores with more ready diffusion of water through them. Conversely, in solutions of lower osmotic pressure, the gel would take up water and its pores become narrow.     

Hydrolysis of soybean oil by lipase with a bioreactor having two different membranes

A membrane bioreactor with 25 sets of two different membranes, one hydrophilic and the other hydrophobic microporous, was used for the hydrolysis of soybean oil by lipase from Candida cylindracea. In the bioreactor, oil, water and enzyme liquid were separately fed into each of 3 clearances between the two membranes. Oil diffused through the hydrophobic membrane and water diffused through the hydrophilic membrane came into contact with the lipase, and hydrolysis subsequently took place. Fatty acid and glycerine produced by the enzymatic reaction diffused through each membrane into the oil and water sides respectively, so that these products were separated from each other. The enzyme was also separated from the products. In this membrane bioreactor, both reaction and separation occurred. Semi-continuous reaction of soybean oil with one cycle of 48 h was carried out for 18 d, at 45°C. Satisfactory performance of the bioreactor, such as a high hydrolysis percentage (92-88%) and enzyme stability over a long-term period was shown. Enzyme stability was found to depend on produced glycerine, as observed by Yamane et al., and the rate of the hydrolysis reaction was controlled by that of the diffusion of fatty acid through the hydrophobic membrane.     

STUDIES ON PERMEABILITY OF MEMBRANES : VIII. THE BEHAVIOR OF THE DRIED COLLODION MEMBRANE TOWARD BIVALENT CATIONS.

A study of the behavior of the dried collodion membrane toward the bivalent calcium ion showed that: 1. There is almost no potential difference established across a membrane separating two calcium chloride solutions of 0.1 and 0.01 N concentrations. 2. The transfer numbers of chlorine and calcium, as measured in electrical transfer experiments, are both close to 0.5. 3. A sample of membrane in equilibrium with a solution of calcium chloride has an extremely high electrical resistance, greater than is observed with solutions of the chlorides of any of the monovalent cations. 4. The total electrolyte content of a membrane in equilibrium with a solution of calcium chloride was only 20 per cent of that observed when the solution was lithium chloride and 10 per cent of that found when the solution was potassium chloride. In explaining these various results it is supposed that (1), (2) and (3) are all the result of (4), that is, of the inability of the calcium ion to penetrate any but the largest of the membrane pores. As the total quantity of electrolyte able to penetrate the membrane is very small the electrical conductivity must also be very small. Moreover, the few larger pores that are large enough to transport the hydrated calcium ion are too large to exert any appreciable effect in decreasing the mobility of the anion. Thus the membrane has no effect in modifying the potentials established across concentration chains with CaCl₂ and the transfer numbers determined experimentally are what one would expect if no membrane were present.     

THE DISTRIBUTION OF EXOGENOUS FERRITIN IN TOAD SPINAL GANGLIA AND THE MECHANISM OF ITS UPTAKE BY NEURONS

Toad spinal ganglion cells are individually enclosed in sheaths consisting of one or more attenuated layers of satellite cell cytoplasm surrounded externally by a basement membrane. Narrow (~150 A) extracellular channels separate these layers from one another and from the underlying neuron. In both in vivo and in vitro experiments it was found that molecules of ferritin, a water-soluble protein, are to some extent able to pass across the basement membrane and through these channels to reach the neuronal plasma membrane. Ferritin particles arriving at the neuronal surface are engulfed by the neuron in 0.1 to 0.2 µ "coated" vesicles. The concentration of ferritin in these vesicles is higher than in the perineuronal space. The ferritin incorporated into the neuron is segregated, apparently intact, in multivesicular bodies. It is inferred that the 150A channels in the satellite cell sheath are patent, aqueous spaces through which molecules with a diameter as large as 95 A are able to pass, and that these neurons are capable of taking up whole protein from their immediate environment by the process of pinocytosis.     

THE CHLOROPHYLL-PROTEIN COMPOUND OF THE GREEN LEAF

1. Aqueous extracts of spinach and Aspidistra leaves yield highly opalescent preparations which are not in true solution. Such extracts differ markedly from colloidal chlorophyll in their spectrum and fluorescence. The differences between the green leaf pigment and chlorophyll in organic solvents are shown to be due to combination of chlorophyll with protein in the leaf. 2. The effect of some agents on extracts of the chlorophyll-protein compound has been investigated. Both strong acid and alkali modify the absorption spectrum, acid converting the compound to the phaeophytin derivative and alkali saponifying the esterified groups of chlorophyll. Even weakly acid solutions (pH 4.5) denature the protein. Heating denatures the protein and modifies the absorption spectrum and fluorescence as earlier described for the intact leaf. The protein is denatured by drying. Low concentrations of alcohol or acetone precipitate and denature the protein; higher concentrations cause dissociation liberating the pigments. 3. Detergents such as digitonin, bile salts, and sodium desoxycholate clarify the leaf extracts but denature the protein changing the spectrum and other properties. 4. Inhibiting agents of photosynthesis are without effect on the absorption spectrum of the chlorophyll-protein compound. 5. The red absorption band of chlorophyll possesses the same extinction value in organic solvents such as ether or petroleum ether, and in aqueous leaf extracts clarified by digitonin although the band positions are different. Using previously determined values of the extinction coefficients of purified chlorophylls a and b, the chlorophyll content of the leaf extracts may be estimated spectrophotometrically. 6. It was found that the average chlorophyll content of the purified chloroplasts was 7.86 per cent. The protein content was 46.5 per cent yielding an average value of 16.1 parts per 100 parts of protein. This corresponds to a chlorophyll content of three molecules of chlorophyll a and one of chlorophyll bfor the Svedberg unit of 17,500. It is suggested that this may represent a definite combining ratio of a and b in the protein molecule.     

Role of pit membranes in macromolecule-induced wilt of plants

Macromolecules present in low concentrations in xylem fluid of Medicago sativa L. var DuPuits will increase the resistance to xylem liquid flow. This increase in resistance was found to be reversible by backflushing the xylem. Autoradiography showed that very large molecules do not pass through pit membrane pores. A comparison of pit membrane pore sizes to molecule sizes suggests that increased resistance to xylem flow is a result of plugging pit membrane pores. It was also found that pit membranes located in two parts of the plant differ in the apparent diameter of their pores and, thus, in their susceptibility to plugging by macromolecules. Macromolecules in xylem fluid may result from hostparasite interactions and may play a significant role in the outcome of the interaction.     

ELECTRON MICROSCOPY OF THE NUCLEAR MEMBRANE OF AMOEBA PROTEUS

An electron microscope study of the nuclear membrane of Amoeba proteus by thin sectioning techniques has revealed an ultrastructure in the outer layer of the membrane that is homologous to the pores and annuli observed in the nuclear membranes of many other cell types studied by these techniques. An inner honeycombed layer apparently unique to Amoeba proteus is also described.     

INFLUENCE OF A SLIGHT MODIFICATION OF THE COLLODION MEMBRANE ON THE SIGN OF THE ELECTRIFICATION OF WATER

1. It is shown that collodion membranes which have received one treatment with a 1 per cent gelatin solution show for a long time (if not permanently) afterwards a different osmotic behavior from collodion membranes not treated with gelatin. This difference shows itself only towards solutions of those electrolytes which have a tendency to induce a negative electrification of the water particles diffusing through the membrane, namely solutions of acids, acid salts, and of salts with trivalent and tetravalent cations; while the osmotic behavior of the two types of membranes towards solutions of salts and alkalies, which induce a positive electrification of the water particles diffusing through the membrane, is the same. 2. When we separate solutions of salts with trivalent cation, e.g. LaCl₃ or AlCl₃, from pure water by a collodion membrane treated with gelatin, water diffuses rapidly into the solution; while no water diffuses into the solution when the collodion membrane has received no gelatin treatment. 3. When we separate solutions of acid from pure water by a membrane previously treated with gelatin, negative osmosis occurs; i.e., practically no water can diffuse into the solution, while the molecules of solution and some water diffuse out. When we separate solutions of acid from pure water by collodion membranes not treated with gelatin, positive osmosis will occur; i.e., water will diffuse rapidly into the solution and the more rapidly the higher the valency of the anion. 4. These differences occur only in that range of concentrations of electrolytes inside of which the forces determining the rate of diffusion of water through the membrane are predominantly electrical; i.e., in concentrations from 0 to about M/16. For higher concentrations of the same electrolytes, where the forces determining the rate of diffusion are molecular, the osmotic behavior of the two types of membranes is essentially the same. 5. The differences in the osmotic behavior of the two types of membranes are not due to differences in the permeability of the membranes for solutes since it is shown that acids diffuse with the same rate through both kinds of membranes. 6. It is shown that the differences in the osmotic behavior of the two types of collodion membranes towards solutions of acids and of salts with trivalent cation are due to the fact that in the presence of these electrolytes water diffuses in the form of negatively charged particles through the membranes previously treated with gelatin, and in the form of positively charged particles through collodion membranes not treated with gelatin. 7. A treatment of the collodion membranes with casein, egg albumin, blood albumin, or edestin affects the behavior of the membrane towards salts with trivalent or tetravalent cations and towards acids in the same way as does a treatment with gelatin; while a treatment of the membranes with peptone prepared from egg albumin, with alanine, or with starch has no such effect.     

STUDIES ON PERMEABILITY OF MEMBRANES : I. INTRODUCTION AND THE DIFFUSION OF IONS ACROSS THE DRIED COLLODION MEMBRANE.

The theoretical aspects of the problem of sieve-like membranes are developed. The method of preparing the dried collodion membrane is described, and the method of defining the property of a particular membrane is given. It consists of the measurement of the Co P, that is the P.D. between an 0.1 and an 0.01 M KCl solution separated by the membrane. Co P is in the best dried membranes 50 to 53 millvolts, the theoretically possible maximum value being 55 millivolts. Diffusion experiments have been carried out with several arrangements, one of which is, for example, the diffusion of 0.1 M KNO₃ against 0.1 M NaCl across the membrane. The amount of K⁺ diffusing after a certain period was in membranes with a sufficiently high Co P (about 50 millivolts or more) on the average ten times as much as the amount of diffused Cl^-. In membranes with a lower Co P the ratio was much smaller, down almost to the proportion of 1:1 which holds for the mobility of these two ions in a free aqueous solution. When higher concentrations were used, e.g. 0.5 M solution, the difference of the rate of diffusion for K⁺ and Cl^- was much smaller even in the best membranes, corresponding to the fact that the P.D. of two KCl solutions whose concentrations are 10:1 is much smaller in higher ranges of concentration than in lower ones. These observations are confirmed by experiments arranged in other ways. It has been shown that, in general, the diffusion of an anion is much slower than the one of a cation across the dried collodion membrane. The ratio of the two diffusion coefficients would be expected to be calculable in connection with the potential difference of such a membrane when interposed between these solutions. The next problem is to show in how far this can be confirmed quantitatively.     

THE PERMEABILITY OF DRY COLLODION MEMBRANES. II

The rate of penetration and the solubility of H, O, N, NH₃, H₂O, HCl gas, CO₂, formic, acetic, chloracetic, dichloracetic acid, glycerol, phenol and mercury bichloride in dry collodion membranes have been measured. The rate of penetration of H and CO₂ is the same whether the membrane and gas are dry or whether the membrane is immersed in water. The solubility of CO₂, acetic acid, phenol and water in collodion is completely reversible and is proportional to the concentration (or vapor pressure) in low concentrations and independent of the surface of the collodion. The size of the pores has been calculated from the vapor pressure of water in the collodion and from the rate of flow of water through the membrane. The results do not agree and are not consistent with the observed rates of penetration. The relative rates of penetration of the gases bear no relation to the density of the gas. When the results are corrected for the solubility of the substances in the collodion and expressed as the diffusion coefficient in collodion they show that the diffusion coefficient increases rapidly as the molecular weight decreases.     

Filtration,diffusion, and molecular sieving through porous cellulose membranes

1. A study has been made of the diffusion and filtration of a graded series of molecules (including tritium-labelled water, urea, glucose, antipyrine, sucrose, raffinose, and hemoglobin) in aqueous solution through porous cellulose membranes of three degrees of porosity. 2. Experimental results were in close agreement with predictions based on the membrane pore theory of Pappenheimer et al. (1,2). Restriction to molecular diffusion is a function of pore radius and molecular radius described by equation (11) in the text. Molecular sieving during ultrafiltration is a function of total pore area per unit path length, pore radius, molecular radius, and filtration rate given by equations (16) and (19). 3. Estimates of average pore radius made by means of this theory were considerably larger than estimates made by the method of Elford and Ferry (3) (Table II). Sources of error in the latter method are discussed and a new method of membrane calibration is proposed in which the total cross-sectional area of the pores is measured by direct diffusion of isotope-labelled water. 4. Steady-state osmotic pressures of solutions of sucrose and raffinose measured during molecular sieving through cellulose membranes were found to be close to the "ideal" osmotic pressures calculated by van't Hoff's law. Thus the present experimental data support the methods used by Pappenheimer et al. in their studies on living capillary walls as well as their theory of membrane pore permeability.     

血红蛋白对人红细胞膜脂质分子侧向运动的影响

用荧光漂白恢复法测定了血红蛋白对红细胞膜脂质分子侧向扩散的限制作用.血红蛋白主要是通过和内侧膜脂质的结合而产生影响的,pH6及PH7.7时都显示出效应的存在.和膜结合较强的高铁血红蛋白,表现出对膜脂质侧向扩散亦有较大的限制作用.     

STUDIES ON THE PERMEABILITY OF MEMBRANES : II. DETERMINATION OF IONIC TRANSFER NUMBERS IN MEMBRANES FROM CONCENTRATION CHAINS.

The ionic transfer number in an electrolyte solution in the pores of a narrow pored collodion membrane depends much more on the concentration than it does in a free aqueous solution. The potential difference of two solutions of the same electrolyte in different concentration depends largely on the concentration range. The ratio of the concentrations on the two sides was always 1:2 in the experiments; the concentration range was varied. It is shown that the transfer number of Cl, calculated from the P.D. measured, is very small in dilute solution (down to .02 and less in some cases), whereas it approaches the value .5 holding for free aqueous solutions when the concentration range is raised. The differences for the transfer number of Cl, according to the cation (H, K, Na, Li), can be recognized and show the same order as in free aqueous solution. But even in LiCl, where in an ordinary aqueous solution the transfer number of Cl is always > .5, this number is very low in the case of the membrane (e.g. < .05 in .01 M solution).     

Potassium-selective amphotericin B channels are predominant in vesicles regardless of sidedness.

Amphotericin B (AmB) is a membrane-active antibiotic which has been shown to increase ion and small molecule permeability in a variety of model and biological membrane systems. A major mechanistic model, based on BLM systems, proposes that amphotericin forms barrellike pores with cholesterol which are cation selective when added to one side of the membrane and anion selective when added to both sides. We have tested this hypothesis on small and reverse-phase large unilamellar vesicles (SUV and REV) with and without cholesterol. The method used to measure K+, Cl-, and net ion currents is based on ion/H+ exchange detected by the entrapped pH probe pyranine. We find that AmB forms channels which have net selectivity for K+ over Cl- regardless of sidedness or sterol content in SUV. REV with 10% cholesterol also show net K+ selectivity with double-sided addition. Differences are noted between cholesterol- and non-sterol-containing vesicles consistent with at least two separate modes of action: (1) cholesterol-containing SUV form some larger diameter pores which allow the passage of larger ions especially when added to both sides; (2) SUV without sterol form pores which are still K+ over Cl- selective, but larger ions do not pass. The latter mode of action precludes a sterol/pore type of model but not necessarily a barrellike model consisting only of amphotericin molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

AMPHOTERIC COLLOIDS : V. THE INFLUENCE OF THE VALENCY OF ANIONS UPON THE PHYSICAL PROPERTIES OF GELATIN.

1. When we plot the values of osmotic pressure, swelling, and viscosity of gelatin solutions as ordinates over the pH as abscissæ, practically identical curves are obtained for the effect of monobasic acids (HCl, HBr, HNO₃, and acetic acid) on these properties. 2. The curves obtained for the effect of H₂SO₄ on gelatin are much lower than those obtained for the effect of monobasic acids, the ratio of maximal osmotic pressures of a 1 per cent solution of gelatin sulfate and gelatin bromide being about 3:8. The same ratio had been found for the ratio of maximal osmotic pressures of calcium and sodium gelatinate. 3. The curves representing the influence of other dibasic and tribasic acids, viz. oxalic, tartaric, succinic, citric, and phosphoric, upon gelatin are almost identical with those representing the effect of monobasic acids. 4. The facts mentioned under (2) and (3) permit us to decide between a purely chemical and a colloidal explanation of the influence of acids on the physical properties of gelatin. In the former case we should be able to prove, first, that twice as many molecules of HBr as of H₂SO₄ combine with a given mass of gelatin; and, second, that the same number of molecules of phosphoric, citric, oxalic, tartaric, and succinic acids as of HNO₃ or HCl combine with the same mass of gelatin. It is shown in the present paper that this is actually the case. 5. It is shown that gelatin sulfate and gelatin bromide solutions of the same pH have practically the same conductivity. This disproves the assumption of colloid chemists that the difference in the effect of bromides and sulfates on the physical properties of gelatin is due to a different ionizing and hydratating effect of the two acids upon the protein molecule.     

MEASUREMENT OF THE RATE OF ACETYLCHOLINE DIFFUSION THROUGH A BRAIN SLICE AND ITS SIGNIFICANCE IN STUDIES OF THE CELLULAR DISTRIBUTION OF ACETYLCHOLINESTERASE

The rate of ACh diffusion through a 0·8 mm thick slice from the surface of the rat cerebral cortex, under aerobic conditions at 37°C, was determined by bathing the intact surface of the slice (compartment A) with ACh containing buffer and determining the concentration of ACh in buffer bathing the cut surface of the slice (compartment B). With 1 or 5 mM-ACh in compartment A no ACh was detectable in compartment B within 3 h unless at least 95 per cent of the AChE, as assessed on homogenates, was inhibited. With a given level of AChE inhibition, the rate of ACh diffusion was dependent on its concentration in compartment A. With 1 mM-ACh in compartment A the difference between the rates of hydrolysis of ACh during diffusion through slices with an AChE inhibition of 98·3 and 99·4 per cent, as assessed by AChE assays of homogenates made from the slices, was only 6 per cent of the difference between the rates of hydrolysis of 1 m-ACh by the homogenates of that part of the slices through which diffusion took place. For 5 mM-ACh and levels of 95 and 99·2 per cent inhibition the corresponding value was 10-3 per cent. Since the concentration of ACh must fall across the slice it is not possible to calculate from these figures the number of enzyme sites involved in the hydrolysis during diffusion, i.e. the concentration of extracellular AChE. The implications of these observations are discussed, particularly in relation to studies of the release of ACh from the cerebral cortex in vivo     

Mechanisms of equinatoxin II-induced transport through the membrane of a giant phospholipid vesicle

Protein equinatoxin II from sea anemone Actinia equina L. was used to form pores in phospholipid membranes. We studied the effect of these pores on the net transmembrane transport of sucrose and glucose by observing single giant (cell-size) vesicles under the phase contrast microscope. Sugar composition in the vesicle was determined by measuring the width of the halo, which appears around the vesicle in the phase contrast image. The transport of sugars was induced when a vesicle, filled with the sucrose solution, was transferred into the isomolar environment of a glucose solution with added equinatoxin II. Typically, a vesicle grew to a critical size, then the membrane broke by bursting and the vesicle shrank, started to grow again, and the whole process was repeated. The consecutive membrane breaks occurred in the same spot. The observed behavior was interpreted by the diffusion flow of the glucose molecules through the equinatoxin II-induced pores and the consequent increase of the vesicle water content. The burst relaxed the critically strained membrane, which then apparently resealed. A mathematical model of the described behavior was developed and was used to obtain the equinatoxin II-induced membrane permeability for the glucose molecules. Its dependence on the equinatoxin II concentration is in agreement with the previous reports.     

THE EFFECT OF HYDROGEN ION CONCENTRATION UPON THE INDUCTION OF POLARITY IN FUCUS EGGS : II. THE EFFECT OF DIFFUSION GRADIENTS BROUGHT ABOUT BY EGGS IN CAPILLARY TUBES

1. When a Fucus egg develops near one end in a close fitting capillary tube of pyrex glass or silica (quartz), diffusion of substances passing to and from the egg is more impeded on the side of the egg toward the far end of the tube. 2. The egg therefore develops in a gradient of its own diffusion products, and of oxygen tension. 3. More than 600 eggs have been reared, each near one end in a capillary, in sea water at various regulated and measured pH values. 4. When the medium, which is initially homogeneous inside and outside the capillary, is initially at pH 6.5 to 7.6, nearly all of the eggs develop rhizoid protuberances on the sides of the eggs toward the far ends of the capillaries. This is on the sides of the eggs where the concentration of substances diffusing from the eggs is greatest. 5. The polarity and developmental pattern of the egg is thus determined either by a concentration gradient of products diffusing from it, or by a gradient of oxygen tension. The former interpretation is favored. 6. This is regarded as an extension of earlier observations that rhizoid protuberances form on the sides of two neighboring eggs in the direction of the neighbor if the sea water is acidified. 7. It appears hardly possible that a mitogenetic effect could be responsible for the response of an egg to its own diffusion gradients. 8. When the medium is made more basic, the percentage of the eggs which form rhizoid protuberances toward the far end of the tube decreases to about 20 or 25 per cent between pH 8.1 and 8.6. The response of the egg to the gradients which it produces is thus statistically reversed. The determination of the polarity of the eggs by the diffusion gradients does not become as complete in alkalinized as in acidified sea water. 9. When the pH of the sea water is elevated to 9.1 or 9.2, salts precipitate out. The type of development is altered and the control of the diffusion gradients over the polarity of the eggs decreases.