Higher permeability for water than for ethyl alcohol in Nitella

If we apply water at one end of a Nitella cell, A, and place at the other end, B, a solution of a substance which does not penetrate, such as sucrose, water enters the cell at A, passes along inside the cell, and escapes at B. But if in place of sucrose we use a substance which penetrates such as ethyl alcohol the flow of water is lessened and this fact makes it possible to measure the amount of alcohol which enters. (An increase in the size of cells placed in solutions of alcohol does not necessarily indicate that the number of mols of alcohol entering is greater than the number of mols of water leaving the cell.) The permeability for water is more than 18 times as great as for ethyl alcohol. The behavior of the 2 substances was compared in the same individual cell with a driving force which at the start was the same for both substances. The number of mols entering per second per cm.² of surface with a driving force of 1 atmosphere at 25°C. is 0.772 (10^–6) for water and 0.042 (10^–6) for ethyl alcohol. The experiments indicate that the non-aqueous substance at the surface of the protoplasm has a higher partition coefficient for water than for ethyl alcohol, although the protoplasmic surface is composed of materials not miscible with water.     

Some aspects of protoplasmic motion

In Nitella the protoplasm forms a layer about 15 microns thick surrounding a large central vacuole. The outer part of the protoplasm is a gel, the inner layer is a sol which is in continual motion travelling the entire length of the cell in opposite directions on opposite sides and thus making a complete circuit (cyclosis). If we have a cell devoid of motion and if we regard the protoplasm in any region as made up of successive portions, A, B, C, D, etc., as we pass from left to right) we may suppose that a reaction starts in B which results in a temporary loss of volume by electrostriction, so that liquid moves from A to B to fill the void thus created. The same reaction then occurs at C causing liquid to flow from B to C and so on. The protoplasmic movement can be controlled by agents which affect the viscosity of the protoplasm or the reactions which cause the flow. Certain reagents such as lead acetate stop the flow temporarily. When the motion is stopped in any region by killing or by applying lead acetate, the motion goes on for a time in adjoining regions. When motion stops in all of the cell or in certain parts, it resumes in the same direction as it had before stoppage occurred. Under normal conditions each of the two sides of the cell (on opposite sides of the white line) has its own characteristic direction of motion which remains unchanged after a temporary stoppage of motion in all parts of the cell. Hence the two sides differ and we have what may be called lateral polarity. There is also longitudinal polarity as the opposite ends of the cell are unlike since shoots grow out at one end and roots at the opposite end. The explanation suggested to account for motion in Nitella may apply to other kinds of motion including the motion of cilia and of flagella.     

Changes in resting potential due to a shift of electrolytes in the cell produced by non-electrolytes

Experiments on Nitella indicate that the resting potential is due chiefly to the outwardly directed diffusion potential of electrolytes which is set up at the inner, non-aqueous, protoplasmic surface surrounding the vacuole. We might therefore expect that any change in the concentration of these electrolytes would affect the resting potential. The experiments described here indicate that this expectation is justified. When a sucrose solution is applied at one end of the cell and water is placed at another spot, water enters at the latter, passes along inside the cell, and escapes into the sucrose solution, but the electrolytes are unable to escape into the sucrose solution (except very slowly) so that the concentration of electrolytes increases in the region in contact with the sucrose solution. Hence the potential at this spot increases. At the other spot where the water enters, the concentration of electrolytes decreases and the potential at this spot falls off. The changes can be carried out reversibly without injury to the cell.     

SOME ASPECTS OF SECRETION : I. SECRETION OF WATER

If we increase the osmotic pressure at one end of a Nitella cell by applying a solution of sucrose and if we subsequently submerge the entire cell in water we find that water enters at the end where the osmotic pressure is higher and comes out of the cell at the other end. If similar inequalities of osmotic pressure should arise as the result of metabolism we can understand how a secreting cell might take up water at one spot on its surface and expel it in another spot and thus bring about the secretion of water. The Nitella cell can expel water from a region of the cell which is in contact with water, air, or mineral oil.     

Transport of water from concentrated to dilute solutions in cells of Nitella

The transport of water from concentrated to dilute solutions which occurs in the kidney and in a variety of living cells presents a problem of fundamental importance. If the cell acts as an osmometer we may expect to bring about such transport by creating an inwardly directed osmotic drive which is higher in one part of the cell than in other regions of the same cell. The osmotic drive is defined as the difference between internal and external osmotic pressure. Experiments with Nitella show that this expectation is justified. If water is placed at one end of the cell (A) and 0.4 M sucrose with an osmotic pressure of 11.2 atmospheres at the other end (B) water enters at A, passes along inside the cell, and escapes at B leaving behind at B the solutes which cannot pass out through the protoplasm. Hence the internal osmotic pressure becomes much higher at B than at A. When 0.4 M sucrose at B is replaced by 0.3 M sucrose with an osmotic pressure of 8.1 atmospheres we find that water enters at B, passes along inside the cell, and escapes at A so that water is transported from a concentrated to a dilute solution although the difference in osmotic pressure of the 2 solutions is more than 8 atmospheres. The solution at B thus becomes more concentrated. It is evident that if metabolism produces a higher osmotic pressure and consequently a higher inwardly directed osmotic drive in one region of the cell as compared with other parts of the same cell water may be transferred from a concentrated to a dilute solution so that the former solution becomes still more concentrated.     

SOME PROPERTIES OF PROTOPLASMIC GELS : II. CONTRACTION OF CHLOROPLASTS IN CURRENTS OF WATER ENTERING THE CELL AND EXPANSION IN OUTGOING CURRENTS

The chloroplasts of Nitella may contract under natural conditions as well as under the influence of certain reagents. When a sufficient amount of water enters any part of the cell they contract in that region and they expand when the direction of the current is reversed. The current may be produced by placing water at one end of the cell and applying at the other end a solution which withdraws water from the cell. The contraction may be due to the washing out of substances from the chloroplast by the ingoing current. The outgoing current bearing dissolved materials from the sap may restore these substances and cause the chloroplasts to resume their normal shape. When blood or sodium dodecylsulfate is present in the ingoing current the contraction of the chloroplast usually fails to occur.     

The mechanism of accumulation in living cells

When a compound enters a living cell until its activity becomes greater inside than outside, it may be said to accumulate. Since it moves from a region where its activity is relatively low to a region where its activity is relatively high, it is evident that work must be done to bring this about. The following explanation is suggested to account for accumulation. The protoplasmic surface is covered with a non-aqueous layer which is permeable to molecules but almost impermeable to ions. Hence free ions cannot enter except in very small numbers. The experiments indicate that ions combine at the outer surface with organic molecules (carrier molecules) and are thus able to enter freely. If upon reaching the aqueous protoplasm these molecules are decomposed or altered so as to set the ions free, the ions must be trapped since they cannot pass out except in very small numbers. If we adopt this point of view we can suggest answers to some important questions. Among these are the following: 1. Why accumulation is confined to electrolytes. This is evident since only ions will be trapped. 2. Why ions appear to penetrate against a gradient. Actually there is no such penetration since the ions enter in combination with molecules. The energy needed to raise the activity of entering compounds is furnished by the reactions involved in the process of accumulation. 3. Why, in absence of injury, ions do not come out when the cell is placed in distilled water. Presumably the outgoing ions will combine at the outer surface with carrier molecules and then move inward in the same way as ions coming from without. 4. Why the relative rate of penetration falls off as the external concentration increases. This is because the entrance of ions is limited by the number of carrier molecules but no such limitation exists when ions move outward since they can do so without combining with carrier molecules. 5. Why accumulation is promoted by constructive metabolism which is needed to build up the organic molecules and by destructive metabolism which brings about their decomposition. 6. Why measuring the mobilities of ions in the outer protoplasmic surface does not enable us to predict the relative rate of entrance of ions. We find for example in Nitella that K⁺ has a much higher mobility than Na⁺ but the accumulation of these ions does not differ greatly. This is to be expected if they enter by combining with molecules at the surface. Only if K⁺ is able to combine preferentially will it accumulate preferentially. 7. Why ions may come out in anoxia and at low temperatures. If these conditions depress the formation of carrier molecules and their decomposition in the protoplasm, the balance between intake and outgo of ions will be disturbed and relatively more may come out. 8. Why the excess of internal over external osmotic pressure is less in sea water than in fresh water. As the external concentration of ions increases the rate of intake does not increase in direct proportion since the number of carrier molecules does not increase and this slows down the relative rate of intake of ions. But it does not slow down the rate of exit of ions since they need not combine with carrier molecules in order to pass out. Hence the excess of ions inside will be relatively less as the concentration of external ions increases. 9. How water is pumped from solutions of higher to solutions of lower osmotic pressure. If metabolism and consequently accumulation is higher at one end of a cell than at the other, the internal osmotic pressure will be higher at the more active end and this makes it possible for the cell to pump water from solutions of higher osmotic pressure at the more active end to solutions of lower osmotic pressure at the less active, as shown experimentally for Nitella. This might help to explain the action of kidney cells and the production of root pressure in plants.     

Movements of water in cells of Nitella

When one end of a Nitella cell (A) is bathed in water and a solution of sucrose is placed at the other (B) we find that water enters at A, travels along inside the cell, and escapes at B. The solutes which cannot pass out through the protoplasm at B remain behind so that the osmotic pressure increases at B and diminishes at A until equilibrium is reached and the motion stops. An equation is given which enables us to predict with considerable accuracy the amount of flow required to produce equilibrium.     

Studies on the electrical properties of a single plant cell; internodal cell of Nitella flexilis

Impedance changes of single plant cells of Nitella flexilis were studied under different environmental conditions. With the analysis presented changes in resistance of the protoplasmic membrane and of cell sap can be studied independently and simultaneously. Under "transcellular osmosis," the resistance of the protoplasmic membrane and of the cell sap increase at the part of the cell where water enters, while they decrease where water goes out. Ethanol of low concentration (below 8 per cent) first decreases and later increases the resistance of the protoplasmic membrane. Concentrated ethanol (over 10 per cent), however, brings about a large decrease in resistance of the protoplasmic membrane. Its time course is not simple, but undulatory changes occur. When ethanol is applied to one part of the cell, the resistance of the protoplasmic membrane shows a different type of change, which may be attributed to the local osmotic effect of ethanol; injury generally occurs with comparatively low concentration. Methanol, ethanol, and propanol have almost the same effect upon the cell, while butanol is toxic at the same concentration. When the cell dies, the resistance of the protoplasmic membrane decreases greatly, while the resistance of the cell sap increases to a level (several hundred kilo ohms or more), expected when external solution and cell sap are freely mixed with each other.     

Abnormal protoplasmic patterns and death in slightly hypertonic solutions

Some interesting properties of protoplasm are revealed when slightly hypertonic solutions of sugars or of electrolytes are applied to Nitella. The chloroplasts contract and the space between them increases and forms a characteristic pattern consisting of clear areas extending lengthwise along the cell and tapering off at both ends. The development of these areas is irreversible from the start. If the cell is returned to water after plasmolysis begins these areas continue to enlarge in much the same fashion as when no change is made in the external solution. The cell soon dies whether returned to water or left in the plasmolyzing solution. Similar results are obtained with other sugars, with NaCl, CaCl(2), and sea water. Similar reactions are also brought about by strong ingoing or outgoing currents of water. This suggests that mechanical action may be chiefly responsible for the result and this idea is in harmony with other facts. It seems possible that the retraction of the protoplasm from the cellulose wall may disturb the delicate non-aqueous film which covers the outer surface of the protoplasm and thus produce injury. Such an effect might take place even without visible retraction if the injury occurred in protoplasmic projections extending into the cellulose wall. A study of this behavior may throw light on the nature of the protoplasmic surface and on the properties of protoplasmic gels as well as on the process of death. An understanding of the mechanism involved may help to explain the action of hypertonic solutions in other cases as, for example, in the artificial parthenogenesis of marine eggs.     

Injury in relation to cell organization

When a part of a Nitella cell, A, is covered with water and the rest of the cell, B, is in contact with a toxic solution there is an escape of solutes at B. This is followed by the escape of solutes at A which causes the death of A. Water enters at A, flows along inside the cell, and escapes at B carrying solutes with it. When this is prevented by covering A with mineral oil the escape of solutes at A is delayed and the life of A is correspondingly prolonged. It is remarkable that this occurs in spite of the fact that the hydrostatic pressure inside the cell (turgor) drops from 6.4 atmospheres to zero. It would seem that A might not be affected by the death of B if the escape of solutes could be prevented.     

The role of water in protoplasmic permeability and in antagonism

The behavior of the cell depends to a large extent on the permeability of the outer non-aqueous surface layer of the protoplasm. This layer is immiscible with water but may be quite permeable to it. It seems possible that a reversible increase or decrease in permeability may be due to a corresponding increase or decrease in the water content of the non-aqueous surface layer. Irreversible increase in permeability need not be due primarily to increase in the water content of the surface layer but may be caused chiefly by changes in the protoplasm on which the surface layer rests. It may include desiccation, precipitation, and other alterations. An artificial cell is described in which the outer protoplasmic surface layer is represented by a layer of guaiacol on one side of which is a solution of KOH + KCl representing the external medium and on the other side is a solution of CO₂ representing the protoplasm. The K⁺ unites with guaiacol and diffuses across to the artificial protoplasm where its concentration becomes higher than in the external solution. The guaiacol molecule thus acts as a carrier molecule which transports K⁺ from the external medium across the protoplasmic surface. The outer part of the protoplasm may contain relatively few potassium ions so that the outwardly directed potential at the outer protoplasmic surface may be small but the inner part of the protoplasm may contain more potassium ions. This may happen when potassium enters in combination with carrier molecules which do not completely dissociate until they reach the vacuole. Injury and recovery from injury may be studied by measuring the movements of water into and out of the cell. Metabolism by producing CO₂ and other acids may lower the pH and cause local shrinkage of the protoplasm which may lead to protoplasmic motion. Antagonism between Na⁺ and Ca⁺⁺ appears to be due to the fact that in solutions of NaCl the surface layer takes up an excessive amount of water and this may be prevented by the addition of suitable amounts of CaCl₂. In Nitella the outer non-aqueous surface layer may be rendered irreversibly permeable by sharply bending the cell without permanent damage to the inner non-aqueous surface layer surrounding the vacuole. The formation of contractile vacuoles may be imitated in non-living systems. An extract of the sperm of the marine worm Nereis which contains a highly surface-active substance can cause the egg to divide. It seems possible that this substance may affect the surface layer of the egg and cause it to take up water. A surface-active substance has been found in all the seminal fluids examined including those of trout, rooster, bull, and man. Duponol which is highly surface-active causes the protoplasm of Spirogyra to take up water and finally dissolve but it can be restored to the gel state by treatment with Lugol solution (KI + I). The transition from gel to sol and back again can be repeated many times in succession. The behavior of water in the surface layer of the protoplasm presents important problems which deserve careful examination.     

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.     

A THEORY OF INJURY AND RECOVERY : II. EXPERIMENTS WITH MIXTURES.

1. The equations which serve to predict the injury of tissue in 0.52 M NaCl and in 0.278 M CaCl₂ and its subsequent recovery (when it is replaced in sea water) also enable us to predict the behavior of tissue in mixtures of these solutions, as well as its recovery in sea water after exposure to mixtures. 2. The reactions which are assumed in order to account for the behavior of the tissue proceed as if they were inhibited by a salt compound formed by the union of NaCl and CaCl₂ with some constituent of the protoplasm (certain of these reactions are accelerated by CaCl₂). 3. In this and preceding papers a quantitative theory is developed in order to explain: (a) the toxicity of NaCl and CaCl₂; (b) the antagonism between these substances; (c) the fact that recovery (in sea water) may be partial or complete, depending on the length of exposure to the toxic solution.     

BIOELECTRIC POTENTIALS IN VALONIA : THE EFFECT OF SUBSTITUTING KCl FOR NaCl IN ARTIFICIAL SEA WATER

The P.D. across the protoplasm of Valonia macrophysa has been studied while the cells were exposed to artificial solutions resembling sea water in which the concentration of KCl was varied from 0 to 0.500 mol per liter. The P.D. across the protoplasm is decreased by lowering and increased by raising the concentration of KCl in the external solution. Changes in P.D. with time when the cell is treated with KCl-rich sea water resemble those observed with cells exposed to Valonia sap. Varying the reaction of natural sea water from pH 5 to pH 10 has no appreciable effect on the P.D. across Valonia protoplasm. Similarly, varying the pH of KCl-rich sea water within these limits does not alter the height of the first maximum in the P.D.-time curve. The subsequent behavior of the P.D., however, is considerably affected by the pH of the KCl-rich sea water. These changes in the shape of the P.D.-time curve have been interpreted as indicating that potassium enters Valonia protoplasm more rapidly from alkaline than from acidified KCl-rich sea water. This conclusion is discussed in relation to certain theories which have been proposed to explain the accumulation of KCl in Valonia sap. The initial rise in P.D. when a Valonia cell is transferred from natural sea water to KCl-rich sea water has been correlated with the concentrations of KCl in the sea waters. It is assumed that the observed P.D. change represents a diffusion potential in the external surface layer of the protoplasm, where the relative mobilities of ions may be supposed to differ greatly from their values in water. Starting with either Planck''s or Henderson''s formula, an equation has been derived which expresses satisfactorily the observed relationship between P.D. change and concentration of KCl. The constants of this equation are interpreted as the relative mobilities of K⁺, Na⁺, and Cl^- in the outer surface layer of the protoplasm. The apparent relative mobility of K⁺ has been calculated by inserting in this equation the values for the relative mobilities of Na⁺ (0.20) and Cl^- (1.00) determined from earlier measurements of concentration effect with natural sea water. The average value for the relative mobility of K⁺ is found to be about 20. The relative mobility may vary considerably among different individual cells, and sometimes also in the same individual under different conditions. Calculation of the observed P.D. changes as phase-boundary potentials proved unsatisfactory.     

PROTOPLASMIC ASYMMETRY IN NITELLA AS SHOWN BY BIOELECTRIC MEASUREMENTS

Using multinucleate cells of Nitella 2 or 3 inches in length it is possible to kill one end with chloroform without producing at the other any immediate alteration which can be detected by our present methods. When a spot in external contact with sap is killed its potential difference falls approximately to zero and it is therefore possible to measure the potential difference across the protoplasm at any desired point merely by leading off from that point to the one where the protoplasm has been killed. The results indicate that the inner and outer protoplasmic surfaces differ, for when both surfaces are in contact with the same solution (cell sap) there is an electromotive force of about 15.9 millivolts, the inner surface being positive to the outer (i.e. the positive current tends to flow from the inner surface through the electrometer to the outer surface). The situation resembles that in Valonia where the corresponding value (with Valonia sap applied to the outside) has been reported as about 14.5 millivolt (the inner surface being positive to the outer). It would seem appropriate to designate this as radial polarity.     

A MICRO INJECTION STUDY ON THE PERMEABILITY OF THE STARFISH EGG

The experiments with the NH₄Cl are similar to, and corroborate micro injection experiments performed in connection with some work on mustard gas in which the writer collaborated. Eggs immersed in sea water containing decomposed mustard gas, at a certain low concentration are not affected. If, however, the solution be injected, the egg quickly cytolyzes owing to the free HCl present. A similar impermeability of the protoplasmic surface film to certain substances was also encountered in injection work on Amœba. Amœbœ immersed in an aqueous solution of eosin will not take the stain till after death. On the other hand, the eosin, when injected into the Amœba, quickly permeates the protoplasm, to be arrested only at the surface. The semipermeability of a living cell appears primarily to be a function of its surface film. It is immaterial whether this film be that of the original cortex of the cell, a film newly formed over a cut surface, or a film that surrounds an artificially induced vacuole within the cell. As long as such a surface film exists neither the acid group of the NH₄Cl nor the alkaline group of the NaHCO₃ can, within certain concentration limits, penetrate the protoplasm. These solutions, if injected beneath the surface film, however, will produce their characteristic effects upon the protoplasm.     

THE KINETICS OF PENETRATION : XX. EFFECT OF pH AND OF LIGHT ON ABSORPTION IN IMPALED HALICYSTIS

The rate of entrance of electrolyte and of water into impaled cells of Halicystis Osterhoutii is unaffected by raising the pH of the sea water to 9.2 or lowering it to 7.0. It is quite possible that sodium enters by combining with an organic acid HX produced by the protoplasm. If the pK'' of this acid is sufficiently low the change in external pH would not produce much effect on the rate of entrance of sodium. The rate of entrance of electrolytes is affected by light. In normal light (i.e. natural succession of daylight and darkness) the rate is about twice as great as in darkness.     

THE EFFECT OF ACETATE BUFFER MIXTURES,ACETIC ACID,AND SODIUM ACETATE,ON THE PROTOPLASM,AS INFLUENCING THE RATE OF PENETRATION OF CRESYL BLUE INTO THE VACUOLE OF NITELLA

When living cells of Nitella are exposed to a solution of sodium acetate and are then placed in a solution of brilliant cresyl blue made up with a borate buffer mixture at pH 7.85, a decrease in the rate of penetration of dye is found, without any change in the pH value of the sap. It is assumed that this inhibiting effect is caused by the action of sodium on the protoplasm. This effect is not manifest if the dye solution is made up with phosphate buffer mixture at pH 7.85. It is assumed that this is due to the presence of a greater concentration of base cations in the phosphate buffer mixture. In the case of cells previously exposed to solutions of acetic acid the rate of penetration of dye decreases with the lowering of the pH value of the sap. This inhibiting effect is assumed to be due chiefly to the action of acetic acid on the protoplasm, provided the pH value of the external acetic acid is not so low as to involve an inhibiting effect on the protoplasm by hydrogen ions as well. It is assumed that the acetic acid either has a specific effect on the protoplasm or enters as undissociated molecules and by subsequent dissociation lowers the pH value of the protoplasm. With acetate buffer mixture the inhibiting effect is due to the action of sodium and acetic acid on the protoplasm. The inhibiting effect of acetic acid and acetate buffer mixture is manifested whether the dye solution is made up with borate or phosphate buffer mixture at pH 7.85. It is assumed that acetic acid in the vacuole serves as a reservoir so that during the experiment the inhibiting effect still persists.     

A nonoccluded virus in nymphs of the dragonfly Leucorrhinia dubia (Odonata,Anisoptera)

This work establishes the sequence of events as the water mold, Coelomomyces psorophorae, develops and enters mosquito larvae through the cuticles of these insects. The fungal cyst shows a number of adaptations for a parasitic function. A bulb-shaped appressorium is produced at germination which is initiated and/or maintained by a microtubular skeleton. The tip of the appressorium secretes a dense amorphous substance, then produces a narrow penetration tube which grows through the larval cuticle. The penetration tube maintains its narrow diameter as it grows inside the host epidermal cell. The parasite protoplasm is injected into a host epidermal cell. The protoplast is squeezed through the narrow tube by the (probably rapid) expansion of a vacuole in the cyst body at the end distal to the penetration point. There is a correlation of cuticular texture with adhesion patterns of cysts. A cuticular collar is always seen in successful penetrations. The sequence of development after attachment of the fungal zygotes is: germination between 1 and 2 hr; penetration between 3 and 4 hr; and injection between 6.5 and 8 hr.