MICRURGICAL STUDIES IN CELL PHYSIOLOGY : I. THE ACTION OF THE CHLORIDES OF NA,K, CA,AND MG ON THE PROTOPLASM OF AM?BA PROTEUS.

By means of micro-dissection and injection Amœba proteus was treated with the chlorides of Na, K, Ca, and Mg alone, in combination, and with variations of pH. I. The Plasmalemma. 1. NaCl weakens and disrupts the surface membrane of the ameba. Tearing the membrane accelerates the disruption which spreads rapidly from the site of the tear. KCl has no disruptive effect on the membrane but renders it adhesive. 2. MgCl₂ and CaCl₂ have no appreciable effect on the integrity of the surface membrane of the ameba when applied on the outside. No spread of disruption occurs when the membrane is torn in these salts. When these salts are introduced into the ameba they render the pellicle of the involved region rigid. II. The Internal Protoplasm. 3. Injected water either diffuses through the protoplasm or becomes localized in a hyaline blister. Large amounts when rapidly injected produce a "rushing effect". 4. HCl at pH 1.8 solidifies the internal protoplasm and at pH 2.2 causes solidification only after several successive injections. The effect of the subsequent injections may be due to the neutralization of the cell-buffers by the first injection. 5. NaCl and KCl increase the fluidity of the internal protoplasm and induce quiescence. 6. CaCl₂ and MgCl₂ to a lesser extent solidify the internal protoplasm. With CaCl₂ the solidification tends to be localized. With MgCl₂ it tends to spread. The injection of CaCl₂ accelerates movement in the regions not solidified whereas the injection of MgCl₂ induces quiescence. III. Pinching-Off Reaction. 7. A hyaline blister produced by the injection of water may be pinched off. The pinched-off blister is a liquid sphere surrounded by a pellicle. 8. Pinching off always takes place with injections of HCl when the injected region is solidified. 9. The injection of CaCl₂ usually results in the pinching off of the portion solidified. The rate of pinching off varies with the concentration of the salt. The injection of MgCl₂ does not cause pinching off. IV. Reparability of Torn Surfaces. 10. The repair of a torn surface takes place readily in distilled water. In the different salt solutions, reparability varies specifically with each salt, with the concentration of the salt, and with the extent of the tear. In NaCl and in KCl repair occurs less readily than in water. In MgCl₂ repair takes place with great difficulty. In CaCl₂ a proper estimate of the process of repair is complicated by the pinching-off phenomenon. However, CaCl₂ is the only salt found to increase the mobility of the plasmalemma, and this presumably enhances its reparability. 11. The repair of the surface is probably a function of the internal protoplasm and depends upon an interaction of the protoplasm with the surrounding medium. V. Permeability. 12. NaCl and KCl readily penetrate the ameba from the exterior. CaCl₂ and MgCl₂ do not. 13. All four salts when injected into an ameba readily diffuse through the internal protoplasm. In the case of CaCl₂ the diffusion may be arrested by the pinching-off process. VI. Toxicity. 14. NaCl and KCl are more toxic to the exterior of the cell than to the interior, and the reverse is true for CaCl₂ and MgCl₂. 15. The relative non-toxicity of injected NaCl to the interior of the ameba is not necessarily due to its diffusion outward from the cell. 16. HCl is much more toxic to the exterior of a cell than to the interior; at pH 5.5 it is toxic to the surface whereas at pH 2.5 it is not toxic to the interior. NaOH to pH 9.8 is not toxic either to the surface or to the interior. VII. Antagonism. 17. The toxic effects of NaCl and of KCl on the exterior of the cell can be antagonized by CaCl₂ and this antagonism occurs at the surface. Although the lethal effect of NaCl is thus antagonized, NaCl still penetrates but at a slower rate than if the ameba were immersed in a solution of this salt alone. 18. NaCl and HCl are mutually antagonistic in the interior of the ameba. No antagonism between the salts and HCl was found on the exterior of the ameba. No antagonism between the salts and NaOH was found on the interior or exterior of the ameba. 19. The pinching-off phenomenon can be antagonized by NaCl or by KCl, and the rate of the retardation of the pinching-off process varies with the concentration of the antagonizing salt. 20. The prevention of repair of a torn membrane by toxic solutions of NaCl or KCl can be antagonized by CaCl₂. These experiments show directly the marked difference between the interior and the exterior of the cell in their behavior toward the chlorides of Na, K, Ca, and Mg.     

THE ORIGIN OF THE POTENTIAL DIFFERENCES RESPONSIBLE FOR ANOMALOUS OSMOSIS

1. Collodion bags coated with gelatin on the inside were filled with a M/256 solution of neutral salt (e.g., NaCl, CaCl₂, CeCl₃, or Na₂SO₄) made up in various concentrations of HNO₃ (varying from N/50,000 to N/100). Each collodion bag was put into an HNO₃ solution of the same concentration as that inside the bag but containing no salt. In this case water diffuses from the outside solution (containing no salt) into the inside solution (containing the salt) with a relative initial velocity which can be expressed by the following rules: (a) Water diffuses into the salt solution as if the particles of water were negatively charged and as if they were attracted by the cation and repelled by the anion of the salt with a force increasing with the valency of the ion. (b) The initial rate of the diffusion of water is a minimum at the hydrogen ion concentration of about N/50,000 HCl (pH 4.7, which is the point at which gelatin is not ionized), rises with increasing hydrogen ion concentration until it reaches a maximum and then diminishes again with a further rise in the initial hydrogen ion concentration. 2. The potential differences between the salt solution and the outside solution (originally free from salt) were measured after the diffusion had been going on for 1 hour; and when these values were plotted as ordinates over the original pH as abscissae, the curves obtained were found to be similar to the osmotic rate curves. This confirms the view expressed by Girard) Bernstein, Bartell, and Freundlich that these cases of anomalous osmosis are in reality cases of electrical endosmose where the driving force is a P.D. between the opposite sides of the membrane. 3. The question arose as to the origin of these P. D. and it was found that the P.D. has apparently a double origin. Certain features of the P.D. curve, such as the rise and fall with varying pH, seem to be the consequence of a Donnan equilibrium which leads to some of the free HNO₃ being forced from the solution containing salt into the outside solution containing no (or less) salt. This difference of the concentration of HNO₃, on the opposite sides of the membrane leads to a P.D. which in conformity with Nernst''s theory of concentration cells should be equal to 58 x (pH inside minus pH outside) millivolts at 18°C. The curves of the values of (pH inside minus pH outside) when plotted as ordinates over the original pH as abscissae lead to curves resembling those for the P. D. in regard to location of minimum and maximum. 4. A second source of the P.D. seems to be diffusion potentials, which exist even if no membranes are present and which seem to be responsible for the fact that the rate of diffusion of negatively charged water into the salt solution increases with the valency of the cation and diminishes with the valency of the anion of the salt. 5. The experiments suggest the possibility that the establishment of a Donnan equilibrium between membrane and solution is one of the factors determining the Helmholtzian electrical double layer, at least in the conditions of our experiments.     

IONIZING INFLUENCE OF SALTS WITH TRIVALENT AND TETRAVALENT IONS ON CRYSTALLINE EGG ALBUMIN AT THE ISOELECTRIC POINT

1. While crystalline egg albumin is highly soluble in water at low temperature at the pH of its isoelectric point, it is coagulated by heating. It has long been known that this coagulation can be prevented by adding either acid or alkali, whereby the protein is ionized. 2. It is shown in this paper that salts with trivalent or tetravalent ions, e.g. LaCl₃ or Na₄Fe(CN)₆, are also able to prevent the heat coagulation of albumin at the isoelectric point (i.e. pH 4.8), while salts with a divalent ion, e.g. CaCl₂, BaCl₄, Na₂SO₄, or salts like NaCl, have no such effect. 3. This is in harmony with the fact shown in a preceding paper that salts with trivalent or tetravalent ions can cause the ionization of proteins at its isoelectric point and thus give rise to a membrane potential between micellæ of isoelectric protein and surrounding aqueous solution, while the above mentioned salts with divalent and monovalent ions have apparently no such effect.     

THE INFLUENCE OF ELECTROLYTES ON THE CATAPHORETIC CHARGE OF COLLOIDAL PARTICLES AND THE STABILITY OF THEIR SUSPENSIONS : II. EXPERIMENTS WITH PARTICLES OF GELATIN,CASEIN, AND DENATURED EGG ALBUMIN.

1. This paper gives measurements of the influence of various electrolytes on the cataphoretic P.D. of particles of collodion coated with gelatin, of particles of casein, and of particles of boiled egg albumin in water at different pH. The influence of the same electrolyte was about the same in all three proteins. 2. It was found that the salts can be divided into two groups according to their effect on the P.D. at the isoelectric point. The salts of the first group including salts of the type of NaCl, CaCl₂, and Na₂SO₄ affect the P.D. of proteins at the isoelectric point but little; the second group includes salts with a trivalent or tetravalent ion such as LaCl₃ or Na₄Fe(CN)₆. These latter salts produce a high P.D. on the isoelectric particles, LaCl₃ making them positively and Na₄Fe(CN)₆ making them negatively charged. This difference in the action of the two groups of salts agrees with the observations on the effect of the same salts on the anomalous osmosis through collodion membranes coated with gelatin. 3. At pH 4.0 the three proteins have a positive cataphoretic charge which is increased by LaCl₃ but not by NaCl or CaCl₂, and which is reversed by Na₄Fe(CN)₆, the latter salt making the cataphoretic charge of the particles strongly negative. 4. At pH 5.8 the protein particles have a negative cataphoretic charge which is strongly increased by Na₄Fe(CN)₆ but practically not at all by Na₂SO₄ or NaCl, and which is reversed by LaCl₃. the latter salt making the cataphoretic charge of the particles strongly positive. 5. The fact that electrolytes affect the cataphoretic P.D. of protein particles in the same way, no matter whether the protein is denatured egg albumin or a genuine protein like gelatin, furnishes proof that the solutions of genuine proteins such as crystalline egg albumin or gelatin are not diaphasic systems, since we shall show in a subsequent paper that proteins insoluble in water, e.g. denatured egg albumin, are precipitated when the cataphoretic P.D. falls below a certain critical value, while water-soluble proteins, e.g. genuine crystalline egg albumin or gelatin, stay in solution even if the P.D. of the particles falls below the critical P.D.     

Attachment and Short-Term Maintenance of Motility and Viability of Entamoeba Histolytica In A Defined Medium*

Organic requirements for attachment to glass, elongation, and motility of Entamoeba histolytica, have been determined. the trophozoite, which has been grown axenically only in highly complex media with reduced oxygen tensions, remains rounded and detached when placed in a Tris-HCl buffered solution containing NaCI, KCI, MgCI₂, and CaCI₂. A maintenance medium in which the amebae could attach to glass, elongate, and remain motile and viable for 12 to 24 h was devised with the addition of cysteine, ascorbic acid, bovine serum albumin, and the vitamin solution of medium NCTC #107. Tris-HCI was the most effective buffer tested and the optimal pH was 6.9 to 7.0. Survival, but not attachment, of the amebae was decreased at osmolalities ranging between 110 and 180 milliosmoles/kg, whereas both functions were decreased above ～260 milliosmoles/kg. Bovine serum albumin, the most effective of the proteins tested, and the vitamin solution helped maintain attachment of some ameba strains, but were not required by other strains. the requirements for cysteine and ascorbic acid were absolute and highly specific. During incubation in the maintenance medium, cell volumes decreased. Sensitivity of the organisms to agglutination by concanavalin A, wheat germ agglutinin, soybean agglutinin and fucose binding protein remained unchanged.     

INFLUENCE OF THE CONCENTRATION OF ELECTROLYTES ON SOME PHYSICAL PROPERTIES OF COLLOIDS AND OF CRYSTALLOIDS

1. When a 1 per cent solution of a metal gelatinate, e.g. Na gelatinate, of pH = 8.4 is separated from distilled water by a collodion membrane, water will diffuse into the solution with a certain rate which can be measured by the rise of the level of the liquid in a manometer. When to such a solution alkali or neutral salt is added the initial rate with which water will diffuse into the solution is diminished and the more so the more alkali or salt is added. This depressing effect of the addition of alkali and neutral salt is greater when the cation of the electrolyte added is bivalent than when it is monovalent. This seems to indicate that the depressing effect is due to the cation of the electrolyte added. 2. When a neutral M/256 solution of a salt with monovalent cation (e.g. Na₂SO₄ or K₄Fe(CN)₆, etc.) is separated from distilled water by a collodion membrane, water will diffuse into the solution with a certain initial rate. When to such a solution alkali or neutral salt is added, the initial rate with which water will diffuse into the solution is diminished and the more so the more alkali or salt is added. The depressing effect of the addition of alkali or neutral salt is greater when the cation of the electrolyte added is bivalent than when it is monovalent. This seems to indicate that the depressing effect is due to the cation of the electrolyte added. The membranes used in these experiments were not treated with gelatin. 3. It can be shown that water diffuses through the collodion membrane in the form of positively charged particles under the conditions mentioned in (1) and (2). In the case of diffusion of water into a neutral solution of a salt with monovalent or bivalent cation the effect of the addition of electrolyte on the rate of diffusion can be explained on the basis of the influence of the ions on the electrification and the rate of diffusion of electrified particles of water. Since the influence of the addition of electrolyte seems to be the same in the case of solutions of metal gelatinate, the question arises whether this influence of the addition of electrolyte cannot also be explained in the same way, and, if this be true, the further question can be raised whether this depressing effect necessarily depends upon the colloidal character of the gelatin solution, or whether we are not dealing in both cases with the same property of matter; namely, the influence of ions on the electrification and rate of diffusion of water through a membrane. 4. It can be shown that the curve representing the influence of the concentration of electrolyte on the initial rate of diffusion of water from solvent into the solution through the membrane is similar to the curve representing the permanent osmotic pressure of the gelatin solution. The question which has been raised in (3) should then apply also to the influence of the concentration of ions upon the osmotic pressure and perhaps other physical properties of gelatin which depend in a similar way upon the concentration of electrolyte added; e.g., swelling. 5. When a 1 per cent solution of a gelatin-acid salt, e.g. gelatin chloride, of pH 3.4 is separated from distilled water by a collodion membrane, water will diffuse into the solution with a certain rate. When to such a solution acid or neutral salt is added—taking care in the latter case that the pH is not altered—the initial rate with which water will diffuse into the solution is diminished and the more so the more acid or salt is added. Water diffuses into a gelatin chloride solution through a collodion membrane in the form of negatively charged particles. 6. When we replace the gelatin-acid salt by a crystalloidal salt, which causes the water to diffuse through the collodion membrane in the form of negatively charged particles, e.g. M/512 Al₂Cl₆, we find that the addition of acid or of neutral salt will diminish the initial rate with which water diffuses into the M/512 solution of Al₂Cl₆, in a similar way as it does in the case of a solution of a gelatin-acid salt.     

ELECTRICAL CHARGES OF COLLOIDAL PARTICLES AND ANOMALOUS OSMOSIS

1. It has been shown in previous publications that when solutions of different concentrations of salts are separated by collodion-gelatin membranes from water, electrical forces participate in addition to osmotic forces in the transport of water from the side of the water to that of the solution. When the hydrogen ion concentration of the salt solution and of the water on the other side of the membrane is the same and if both are on the acid side of the isoelectric point of gelatin (e.g. pH 3.0), the electrical transport of water increases with the valency of the cation and inversely with the valency of the anion of the salt in solution. Moreover, the electrical transport of water increases at first with increasing concentration of the solution until a maximum is reached at a concentration of about M/32, when upon further increase of the concentration of the salt solution the transport diminishes until a concentration of about M/4 is reached, when a second rise begins, which is exclusively or preeminently the expression of osmotic forces and therefore needs no further discussion. 2. It is shown that the increase in the height of the transport curves with increase in the valency of the cation and inversely with the increase in the valency of the anion is due to the influence of the salt on the P.D. (E) across the membrane, the positive charge of the solution increasing in the same way with the valency of the ions mentioned. This effect on the P.D. increases with increasing concentration of the solution and is partly, if not essentially, the result of diffusion potentials. 3. The drop in the transport curves is, however, due to the influence of the salts on the P.D. (ε) between the liquid inside the pores of the gelatin membrane and the gelatin walls of the pores. According to the Donnan equilibrium the liquid inside the pores must be negatively charged at pH 3.0 and this charge is diminished the higher the concentration of the salt. Since the electrical transport is in proportion to the product of E x ε and since the augmenting action of the salt on E begins at lower concentrations than the depressing action on ε, it follows that the electrical transport of water must at first rise with increasing concentration of the salt and then drop. 4. If the Donnan equilibrium is the sole cause for the P.D. (ε) between solid gelatin and watery solution the transport of water through collodion-gelatin membranes from water to salt solution should be determined purely by osmotic forces when water, gelatin, and salt solution have the hydrogen ion concentration of the isoelectric point of gelatin (pH = 4.7). It is shown that this is practically the case when solutions of LiCl, NaCl, KCl, MgCl₂, CaCl₂, BaCl₂, Na₂SO₄, MgSO₄ are separated by collodion-gelatin membranes from water; that, however, when the salt has a trivalent (or tetravalent?) cation or a tetravalent anion a P.D. between solid isoelectric gelatin and water is produced in which the wall assumes the sign of charge of the polyvalent ion. 5. It is suggested that the salts with trivalent cation, e.g. Ce(NO₃)₃, form loose compounds with isoelectric gelatin which dissociate electrolytically into positively charged complex gelatin-Ce ions and negatively charged NO₃ ions, and that the salts of Na₄Fe(CN)₆ form loose compounds with isoelectric gelatin which dissociate electrolytically into negatively charged complex gelatin-Fe(CN)₆ ions and positively charged Na ions. The Donnan equilibrium resulting from this ionization would in that case be the cause of the charge of the membrane.     

DONNAN EQUILIBRIUM AND THE PHYSICAL PROPERTIES OF PROTEINS : II. OSMOTIC PRESSURE.

1. It had been shown in previous publications that the osmotic pressure of a 1 per cent solution of a protein-acid salt varies in a characteristic way with the hydrogen ion concentration of the solution, the osmotic pressure having a minimum at the isoelectric point, rising steeply with a decrease in pH until a maximum is reached at pH of 3.4 or 3.5 (in the case of gelatin and crystalline egg albumin), this maximum being followed by a steep drop in the osmotic pressure with a further decrease in the pH of the gelatin or albumin solution. In this paper it is shown that (aside from two minor discrepancies) we can calculate this effect of the pH on the osmotic pressure of a protein-acid salt by assuming that the pH effect is due to that unequal distribution of crystalloidal ions (in particular free acid) on both sides of the membrane which Donnan''s theory of membrane equilibrium demands. 2. It had been shown in preceding papers that only the valency but not the nature of the ion (aside from its valency) with which a protein is in combination has any effect upon the osmotic pressure of the solution of the protein; and that the osmotic pressure of a gelatin-acid salt with a monovalent anion (e.g. Cl, NO₃, acetate, H₂PO₄, HC₂O₄, etc.) is about twice or perhaps a trifle more than twice as high as the osmotic pressure of gelatin sulfate where the anion is bivalent; assuming that the pH and gelatin concentrations of all the solutions are the same. It is shown in this paper that we can calculate with a fair degree of accuracy this valency effect on the assumption that it is due to the influence of the valency of the anion of a gelatin-acid salt on that relative distribution of the free acid on both sides of the membrane which Donnan''s theory of membrane equilibrium demands. 3. The curves of the observed values of the osmotic pressure show two constant minor deviations from the curves of the calculated osmotic pressure. One of these deviations consists in the fact that the values of the ascending branch of the calculated curves are lower than the corresponding values in the curves for the observed osmotic pressure, and the other deviation consists in the fact that the drop in the curves of calculated values occurs at a lower pH than the drop in the curves of the observed values.     

Does progressive nuclear staining with hemalum (alum hematoxylin) involve DNA,and what is the nature of the dye-chromatin complex?

Previous investigators have disagreed about whether hemalum stains DNA or its associated nucleoproteins. I review here the literature and describe new experiments in an attempt to resolve the controversy. Hemalum solutions, which contain aluminum ions and hematein, are routinely used to stain nuclei. A solution containing 16 Al³⁺ ions for each hematein molecule, at pH 2.0–2.5, provides selective progressive staining of chromatin without cytoplasmic or extracellular “background color.” Such solutions contain a red cationic dye-metal complex and an excess of Al³⁺ ions. The red complex is converted to an insoluble blue compound, assumed to be polymeric, but of undetermined composition, when stained sections are blued in water at pH 5.5–8.5. Staining experiments with DNA, histone and DNA + histone mixtures support the theory that DNA, not histone, is progressively colored by hemalum. Extraction of nucleic acids, by either a strong acid or nucleases at near neutral pH, prevented chromatin staining by a simple cationic dye, thionine, pH 4, and by hemalum, with pH adjustments in the range, 2.0–3.5. Staining by hemalum at pH 2.0–3.5 was not inhibited by methylation, which completely prevented staining by thionine at pH 4. Staining by hemalum and other dye-metal complexes at pH ≤ 2 may be due to the high acidity of DNA-phosphodiester (pK_a ~ 1). This argument does not explain the requirement for a much higher pH to stain DNA with those dyes and fluorochromes not used as dye-metal complexes. Sequential treatment of sections with Al₂(SO₄)₃ followed by hematein provides nuclear staining that is weaker than that attainable with hemalum. Stronger staining is seen if the pH is raised to 3.0–3.5, but there is also coloration of cytoplasm and other materials. These observations do not support the theory that Al³⁺ forms bridges between chromatin and hematein. When staining with hematein is followed by an Al₂(SO₄)₃ solution, there is no significant staining. Taken together, the results of my study indicate that the red hemalum cation is electrostatically attracted to the phosphate anion of DNA. The bulky complex cation is too large to intercalate between base pairs of DNA and is unlikely to fit into the minor groove. The short range van der Waals forces that bind planar dye cations to DNA probably do not contribute to the stability of progressive hemalum staining. The red cation is precipitated in situ as a blue compound, insoluble in water, ethanol and water-ethanol mixtures, when a stained preparation is blued at pH > 5.5.     

A STUDY OF PHAGOCYTOSIS IN THE AMEBA CHAOS CHAOS

The process of phagocytosis was investigated by observing the interactions between the ameba Chaos chaos and its prey (Paramecium aurelia), by studying food cup formation in the living cell, and by studying the fine structure of the newly formed cup using electron microscopy of serial sections. The cytoplasm surrounding the food cup was found to contain structures not seen elsewhere in the ameba. The results are discussed in relation to the mechanisms which operate during food cup formation.     

ION SERIES AND THE PHYSICAL PROPERTIES OF PROTEINS. I

1. This paper contains experiments on the influence of acids and alkalies on the osmotic pressure of solutions of crystalline egg albumin and of gelatin, and on the viscosity of solutions of gelatin. 2. It was found in all cases that there is no difference in the effects of HCl, HBr, HNO₃, acetic, mono-, di-, and trichloracetic, succinic, tartaric, citric, and phosphoric acids upon these physical properties when the solutions of the protein with these different acids have the same pH and the same concentration of originally isoelectric protein. 3. It was possible to show that in all the protein-acid salts named the anion in combination with the protein is monovalent. 4. The strong dibasic acid H₂SO₄ forms protein-acid salts with a divalent anion SO₄ and the solutions of protein sulfate have an osmotic pressure and a viscosity of only half or less than that of a protein chloride solution of the same pH and the same concentration of originally isoelectric protein. Oxalic acid behaves essentially like a weak dibasic acid though it seems that a small part of the acid combines with the protein in the form of divalent anions. 5. It was found that the osmotic pressure and viscosity of solutions of Li, Na, K, and NH₄ salts of a protein are the same at the same pH and the same concentration of originally isoelectric protein. 6. Ca(OH)₂ and Ba(OH)₂ form salts with proteins in which the cation is divalent and the osmotic pressure and viscosity of solutions of these two metal proteinates are only one-half or less than half of that of Na proteinate of the same pH and the same concentration of originally isoelectric gelatin. 7. These results exclude the possibility of expressing the effect of different acids and alkalies on the osmotic pressure of solutions of gelatin and egg albumin and on the viscosity of solutions of gelatin in the form of ion series. The different results of former workers were probably chiefly due to the fact that the effects of acids and alkalies on these proteins were compared for the same quantity of acid and alkali instead of for the same pH.     

PH-DEPENDENCE OF CHLORTETRACYCLINE(CTC)-INDUCED PLUG FORMATION IN NITELLA FLEXILIS (CHARACEAE)1

The formation of chlortetracycline(CTC)-induced wall appositions (callose plugs) in Nitella flexilis (L.)Ag. was pH-dependent in the range between 4.3-8.3. Plug number and plug diameter increased with the pH of the CTC solution. At pH 4.3 plug formation was light-dependent and occurred below the alkaline regions of the cell surface which form during photo synthetic assimilation of HCO₃^?. Inhibition of photosynthesis by 3–(3′,4′-dichlorophenyl)-1, 1-dimethylurea prevented plug formation in the light. Dark-treated cells could be induced to form plugs by raising the pH of the CTC solution. The formation of large but incomplete plugs in the presence of cytochalasin B is explained by the formation of numerous weak alkaline sites. I suggest that CTC enhances locally the Ca²⁺content at the cytoplasm near the plasmamembrane. The ionophoric character of CTC is probably more pronounced at high pH mainly because of a weaker binding with cations and a closer contact with the membrane.     

THE PENETRATION OF AMMONIA INTO FROG MUSCLE

1. A study was made of the electrolyte changes which occur when frog muscles are immersed in a Ringer solution with 1/5 of the Na replaced by NH₄Cl. Analyses were made in the solution and in the muscles for K and NH₃, and the muscles were also analyzed for Cl, HCO₃, and Na. Control muscles were immersed in normal Ringer''s solution and similarly analyzed. 2. The amount of ammonia taken up was about equal to the K and Na lost. There was also a small increase in chloride content. The bicarbonate content was the same in both experimental and control muscles, indicating no change in the muscle pH due to the NH₃ which penetrated. An increased loss of K due to the penetration of NH₃ was also demonstrated by the use of radioactive K. 3. After 5 hours, the concentration of ammonia per gram of muscle is about the same as the concentration in the solution. After 4 or 5 days, the concentration in the muscle is about 1.5 times that in the solution. The inside to outside NH₃ ratio is about equal to the corresponding H ion ratio, but is much less than the K ratio. 4. The rate of penetration of the NH₃ is increased by a rise of temperature, by stirring the solution, and by decrease in the concentration of Na, K, Ca, or Mg in the solution; it is decreased by increasing the size of the muscles or by killing them with chloroform or boiling. 5. Liver, smooth muscle, skin, and kidney, in a few experiments, behaved much like muscle except that there was a formation of urea in the case of liver. 6. The injection of NH₄Cl into anesthetized cats causes an increase in the level of K in the blood plasma.     

THE PENETRATION OF BASIC DYE INTO NITELLA AND VALONIA IN THE PRESENCE OF CERTAIN ACIDS,BUFFER MIXTURES,AND SALTS

When living cells of Nitella are exposed to an acetate buffer solution until the pH value of the sap is decreased and subsequently placed in a solution of brilliant cresyl blue, the rate of penetration of dye into the vacuole is found to decrease in the majority of cases, and increase in other cases, as compared with the control cells which are transferred to the dye solution directly from tap water. This decrease in the rate is not due to the lowering of the pH value of the solution just outside the cell wall, as a result of diffusion of acetic acid from the cell when cells are removed from the buffer solution and placed in the dye solution, because the relative amount of decrease (as compared with the control) is the same whether the external solution is stirred or not. Such a decrease in the rate may be brought about without a change in the pH value of the sap if the cells are placed in the dye solution after exposure to a phosphate buffer solution in which the pH value of the sap remains normal. The rate of penetration of dye is then found to decrease. The extent of this decrease is the greater the lower the pH value of the solution. It is found that hydrochloric acid and boric acid have no effect while phosphoric acid has an inhibiting effect at pH 4.8 on stirring. Experiments with neutral salt solutions indicate that a direct effect on the cell (decreasing penetration) is due to monovalent base cations, while there is no such effect directly on the dye. It is assumed that the effect of the phosphate and acetate buffer solutions on the cell, decreasing the rate of penetration, is due (1) to the penetration of these acids into the protoplasm as undissociated molecules, which dissociate upon entrance and lower the pH value of the protoplasm or to their action on the surface of the protoplasm, (2) to the effect of base cations on the protoplasm (either at the surface or in the interior), and (3) possibly to the effect of certain anions. In this case the action of the buffer solution is not due to its hydrogen ions. In the case of living cells of Valonia under the same experimental conditions as Nitella it is found that the rate of penetration of dye decreases when the pH value of the sap increases in presence of NH₃, and also when the pH value of the sap is decreased in the presence of acetic acid. Such a decrease may be brought about even when the cells are previously exposed to sea water containing HCl, in which the pH value of the sap remains normal.     

Potentiometric measurements of carbon dioxide flux of submerged aquatic macrophytes in pH-statted natural waters

SUMMARY. 1, An apparatus has been described that is suitable for potentiometric measurement of carbon dioxide flux in photosynthesizing shoots of submerged aquatic macrophytes 2. The procedure, based on methods described by Tailing (1973) for measurement of phytoplankton photosynthesis, relies upon the continuous pH-statting of the solution surrounding the tissues. The pH of the solution is monitored by electrodes from a pH meter which is linked to an auto-titrator. The rise in pH during photosynthesis is then compensated tor by controlled, small titrant additions of CO₂-ennched solution (titrant water). This replaces the CO₂ removed by the tissues without affecting the total alkalinity of the solution. If the concentration of CO₂ in the titrant water, and the volume of titrant added arc known precisely, the CO₂ flux can be calculated. 3. Total alkalinity, total CO₂ and free-CO₂ acidity of the bathing solutions and titrant waters are estimated by Gran titrations and the pH: tilre-volume data pairs are analysed by computer to provide rapid data feed-back. A modification to Tailing's equation for calculation of F₁functions has been necessary for accurate calibration of the CO₂enriched tilrant water. 4. The photosynthesis cuvette, which is surrounded by a water-jacket, is approximately I dm³ in capacity and has six compartments for the shoots. An impeller at the base of the cuvette rapidly mixes and cycles the bathing solution and flushes it over the tissues. 5. Information on temperature, light flux density, oxygen concentration. pH and titre-volume is continuously recorded into a data-logger and is fed into a computer which is programmed for data analyses. 6. Results from a typical experiment show the system to be sound and the method has considerable potential, especially in the study of aquatic plant photosynthesis in natural waters.     

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.     

Infrared spectroscopic studies of aqueous solutions of dioxouranium(VI) and its hydrolysed products and of in situ electro-generated dioxouranium(V)

Aqueous solutions of dioxouranium(VI) (pH range 0 to 4) give rise to bands at 954 and 938 cm⁻¹ attributable to the v₃(MO₂) stretching modes of the UO₂²⁺ and (UO₂)₂(OH)_2²⁺ cations, respectively. A shoulder at 916 cm⁻¹ is assigned to the v₃(MO₂) mode of hydrolysed dioxouranium(VI) species of higher nuclearity. Infrared spectro-electrochemical studies using a thin-layer reflection-absorption cell have facilitated the study of the reduction of aqueous solutions of dioxouranium(VI) to yield dioxouranium(V) which may be further reduced to uranium(IV). The electrogeneration of dioxouranium(V) is monitored by following the increase in intensity of a band at 914 cm⁻¹ which is present in the spectra at potentials between −0.2 and −0.8 V. The dioxouranium(V) species is predominantly in the form UO₂⁺, which may be in solution or incorporated into an insoluble phase of uranium oxides which deposit onto the working electrode. The U^VO bond length is estimated to be 1.76 Å, 0.03 Å longer than the U^VIO bond in aqueous solution. The maximum concentration of UO₂⁺able to be achieved is highly dependent on the pH and is optimum at pH 3.4. Changes in the pH of the solution under study can be monitored by infrared spectroscopy during the course of the reduction by determining the relative concentrations of hydrolysed dioxouranium(VI) species.     

Sulfur-dioxide fluxes into different cellular compartments of leaves photosynthesizing in a polluted atmosphere

A computer model is used to analyze fluxes of SO₂ from polluted air into leaves and the intracellular distribution of sulfur species derived from SO₂. The analysis considers only effects of acidification and of anion accumulation. (i) The SO₂ flux into leaves is practically exclusively controlled by the boundary-layer resistance of leaves to gas diffusion and by stomatal opening. At constant stomatal opening, flux is proportional to the concentration of SO₂ in air. (ii) The sink capacity of cellular compartments for SO₂ depends on intracellular pH and the intracellular localization of reactions capable of oxidizing or reducing SO₂. In the mesophyll of illuminated leaves, the chloroplasts possess the highest trapping potential for SO₂. (iii) If intracellular ion transport were insignificant, and if bisulfite and sulfite could not be oxidized or reduced, leaves with opened stomata would rapidly be killed both by the accumulation of sulfites and by acidification of chloroplasts and cytosol even if SO₂ levels in air did not exceed concentrations thought to be permissible. Acidification and sulfite accumulation would remain confined largely to the chloroplasts and to the cytosol under these conditions. (iv) Transport of bisulfite and protons produced by hydration of SO₂ into the vacuole cannot solve the problem of cytoplasmic accumulation of bisulfite and sulfite and of cytoplasmic acidification, because SO₂ generated in the acidic vacuole from the bisulfite anion would diffuse back into the cytoplasm. (v) Oxidation to sulfate which is known to occur mainly in the chloroplasts can solve the problem of cytoplasmic sulfite and bisulfite accumulation, but aggravates the problem of chloroplastic and cytosolic acidification. (vi) A temporary solution to the problem of acidification requires the transfer of H⁺ and sulfate into the vacuole. This transport needs to be energized. The storage capacity of the vacuole for protons and sulfate defines the extent to which SO₂ can be detoxified by oxidation and removal of the resulting protons and sulfate anions from the cytoplasm. Calculations show that even at atmospheric levels of SO₂ thought to be tolerable, known vacuolar buffer capacities are insufficient to cope with proton production during oxidation of SO₂ to sulfate within a vegetation period. (vii) A permanent solution to the problem of acidification is the removal of protons. Protons are consumed during the reduction of sulfate to sulfide. Proteins and peptides contain sulfur at the level of sulfide. During photosynthesis in the presence of the permissible concentration of 0.05l·l^-1 SO₂, sulfur may be deposited in plants at a ratio not far from 1/500 in relation to carbon. The content of reduced sulfur to carbon is similar to that ratio only in fast-growing, protein-rich plants. Such plants may experience little difficulty in detoxifying SO₂. In contrast, many trees may contain reduced sulfur at a ratio as low as 1/10 000 in relation to carbon. Excess sulfur deposited in such trees during photosynthesis in polluted air gives rise to sulfate and protons. If detoxification of SO₂ by reduction is inadequate, and if the storage capacity of the vacuoles for protons and sulfate is exhausted, damage is unavoidable. Calculations indicate that trees with a low ratio of reduced S to C cannot tolerate long-term exposure to concentrations of SO₂ as low as 0.02 or 0.03 l·l^-1 which so far have been considered to be non-toxic to sensitive plant species.     

Cell Size,Macromolecular Composition,and O2 Consumption During Agitated Cultivation of Naegleria gruberi*

SYNOPSIS. Cell size, macromolecular composition, carbohydrate utilization patterns, and O₂ concentrations were measured throughout the growth stages of Naegleria gruberi in agitated cultures in a complex medium. Biphasic logarithmic growth occurred during the initial 83 hr of growth and the mean generation time was 7.0 hr and 19 hr during initial and secondary log growth stages, respectively. The maximum yield was 5 × 10* amebaeJml. The pH rose rapidly (1 pH unit) during the secondary log growth phase (52-83 hr) and continued into the stationary growth phase (83-120 hr). Dry weight, total protein, carbohydrate, and RNA per ameba increased just before the secondary log growth phase. RNA increased 31% to 35% per ameba at the end of each phase of log growth. DNA increased ～ 2-fold throughout the different growth phases. Average cell size increased 90% during biphasic log growth then decreased during stationary phase. O₂ tension decreased from 100% to 18% of saturation during the biphasic growth phase, then increased during stationary growth to near 100% saturation. Glucose and total carbohydrate assays showed little utilization of those substrates throughout the growth stages. Naegleria gruberi presumably has a predominantly aerobic metabolism, also its metabolism may change during the different growth phases.     

STUDIES ON THE REGULATION OF OSMOTIC PRESSURE : II. THE EFFECT OF INCREASING CONCENTRATIONS OF ALBUMIN ON THE CONDUCTIVITY OF A SODIUM CHLORIDE SOLUTION.

Egg albumin, like gelatin, influences the conductivity of a 0.6 per cent NaCl solution in two ways: (a) At an hydrogen ion concentration of about pH 3.0, increasing concentrations increase the conductivity. (b) Near the isoelectric point of albumin and at the pH of the blood, increasing concentrations of albumin decrease the conductivity of the NaCl solution.