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1. Permeability to water in unfertilized eggs of the sea urchin, Arbacia punctulata, is found to be greater in hypotonic solutions of dextrose, saccharose and glycocoll than in sea water of the same osmotic pressure. 2. The addition to dextrose solution of small amounts of CaCl(2) or MgCl(2) restores the permeability approximately to the value obtained in sea water. 3. This effect of CaCl(2) and MgCl(2) is antagonized by the further addition of NaCl or KCl. 4. It is concluded that the NaCl and KCl tend to increase the permeability of the cell to water, CaCl(2) and MgCl(2) to decrease it. 5. The method here employed can be used for quantitative study of salt antagonism.

Rabbits infected intravenously with virulent mammalian tubercle bacilli have in a majority of cases developed circulating antibodies to a slight but appreciable degree. The increase in titer was detected in one group of rabbits within the 2nd or 3rd week, in others during the 2nd month of infection. Rabbits with residual pulmonary foci, resulting from infection 6 months or more previously with human tubercle bacilli, on reinfection with bovine tubercle bacilli promptly developed circulating antibodies strikingly in excess of those found during the course of primary infection. Such antibodies were present 6 or 7 days after reinfection. The changes in titer during tuberculous infection as detected by the bacterial surface reactions and by phagocytosis were again, within the experimental error, in quantitative correspondence. The loss of phagocytosis-promoting power in heated normal serum involves an exception to this correspondence between surface and phagocytosis effects. This exception has already been discussed in an earlier paper (8).

1. Solutions containing NH(4)OH and NaOH, and CO(2) and HCl may be used to produce various combinations of extracellular and intracellular reactions in starfish eggs, Gonionemus, and Nitella cells. 2. Staining by basic dyes is, with a constant intracellular reaction, favored by increased extracellular alkalinity. With a constant extracellular reaction, staining is hindered by increased intracellular alkalinity. 3. These facts are in opposition to the view that staining of cells by basic dyes is chiefly governed by a combination of the dyes with cell proteins. It is more in harmony with the view that the combination is with a substance or substances of acid nature.

The rate of swelling of unfertilized sea urchin eggs in hypotonic sea water was investigated. Analysis of curves leads to the following conclusions. 1. The rate of swelling follows the equation, See PDF for Equation where V(eq)., V(0), and V(t) stand for volume at equilibrium, at first instant, and at time t, respectively, the other symbols having their usual significance. This equation is found to hold over a wide range of temperatures and osmotic pressures. This relation is the one expected in a diffusion process. 2. The rate of swelling is found to have a high temperature coefficient (Q(10) = 2 to 3, or micro = 13,000 to 19,000). This deviation from the usual effect of temperature on diffusion processes is thought to be associated with changes in cell permeability to water. The possible influence of changes in viscosity is discussed. 3. The lower the osmotic pressure of the solution, the longer it takes for swelling of the cell. Thus at 15 degrees in 80 per cent sea water, the velocity constant has a value of 0.072, in 20 per cent sea water, of 0.006.

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.

1. Permeability to water in unfertilized eggs of the sea urchin, Arbacia punctulata, was studied by measuring the rate of swelling in hypotonic dextrose solution. 2. Permeability is greatly affected by addition of electrolytes in low concentration. 3. A decrease in permeability to water was found with increasing valence of the cation, using a series of cobaltammine chlorides in which the valence of the cation ranged from 1 to 6. 4. Conversely, an increase in permeability to water was found with increasing valence of the anion, using two series of potassium salts in which the valence of the anion ranged from 1 to 4, and 1 to 3, respectively. 5. It is concluded that the effect of electrolytes on permeability to water depends chiefly on the sign and the number of charges on the ion, in the sense that positive ions decrease permeability to water, while negative ions increase permeability to water; and the effectiveness of the ion is greater the higher its valence. 6. Antagonism has been demonstrated between cations and anions in their effect on permeability, and the method employed permits quantitative study of such antagonism.

Using unfertilized eggs of Arbacia punctulata as natural osmometers an attempt has been made to account for the course of swelling and shrinking of these cells in anisotonic solutions by means of the laws governing osmosis and diffusion. The method employed has been to compute permeability of the cell to water, as measured by the rate of volume change per unit of cell surface per unit of osmotic pressure outstanding between the cell and its medium. Permeability to water as here defined and as somewhat differently defined by Northrop is approximately constant during swelling and shrinking, at least for the first several minutes of these processes. Permeability is found to be independent of the osmotic pressure of the solution in which cells are swelling. Water is found to leave cells more readily than it enters, that is, permeability is greater during exosmosis than during endosmosis.

1. The rate of exosmosis of water was studied in unfertilized Arbacia eggs, in order to bring out possible differences between the kinetics of exosmosis and endosmosis. 2. Exosmosis, like endosmosis, is found to follow the equation See PDF for Equation, in which a is the total volume of water that will leave the cell before osmotic equilibrium is attained, x is the volume that has already left the cell at time t, and k is the velocity constant. 3. The velocity constants of the two processes are equal, provided the salt concentration of the medium is the same. 4. The temperature characteristic of exosmosis, as of endomosis, is high. 5. It is concluded that the kinetics of exosmosis and endosmosis of water in these cells are identical, the only difference in the processes being in the direction of the driving force of osmotic pressure.

WE HAVE ATTEMPTED TO ANSWER THE QUESTION: How nearly ideal, as an osmometer, is the unfertilized Arbacia egg? The following conclusion have been reached: 1. Volumes can be measured accurately over a wide range of pressures since the cell is in general spherical and does not suffer deformation from its own weight or other factors. 2. The product of volume and pressure is approximately constant, if allowance be made for osmotically inactive cell contents. It is computed that from 7 to 14 per cent of cell volume is occupied by osmotically inactive material. 3. Evidence is presented that no appreciable escape of cell contents occurs while the cell is in hypotonic sea water; that, therefore, the semipermeability of the membrane is approximately perfect, so long as injury to the cell is avoided. 4. In comparison with osmotic pressure the influence of other forces, such as elasticity or surface tension, on cell volume must in these experiments be slight.

Methods are described for investigating the relation between phagocytosis of bacteria by polymorphonuclear leucocytes, and certain physical-chemical properties of the bacterial surface. Serum sensitization causes the following changes in the properties of acid-fast bacteria: (a) increased cohesiveness, (b) decrease in surface electric potential difference, (c) decrease in wettability of the bacteria by oil, and (d) increased phagocytosis. Tests have been conducted periodically with the sera of 4 rabbits under active immunization with as many strains of acid-fast bacteria; the parallelism between the alteration in bacterial surface properties and the promotion of phagocytosis by these sera has been, within the experimental error, complete. The percentage of phagocytosis of a given bacterial suspension has been found to depend both upon the sensitizing serum component or components deposited upon the bacterium and upon the intrinsic properties of the unsensitized bacterial surface.

As a further test of the theory of tropin action proposed in the preceding paper artificial surfaces have been prepared, and have been found to be phagocytized according to prediction from the theory. Protein was adsorbed on collodion particles according to the technique of F. S. Jones. These particles were then agglutinated and prepared for phagocytosis by the corresponding protein precipitin sera. The precipitating, agglutinating, surface and tropin effects for each serum or serum globulin fraction have been found to be in satisfactory quantitative correspondence. All of these effects were serologically specific; all remained almost unaffected by inactivation of the immune sera for 30 minutes at 56 degrees C. or by washing of the particles after sensitization. The surfaces of particles maximally sensitized by homologous rabbit immune serum or one of its globulin fractions have shown certain characteristic properties, i.e., they were cohesive, had wetting properties characteristic for protein, and were isoelectric at pH. 5.5 to 5.8. The same set of properties were found for immune precipitate in the zone of maximal precipitation. The same properties have also been found for maximally sensitized acid-fast bacteria, and for maximally sensitized sheep erythrocytes. These results indicate, we believe, that precipitation, agglutination, the surface changes and increased phagocytosis are all consequences of one underlying phenomenon. This phenomenon is the specific chemcal combination with, and deposit on the surface of the antigen of antibody protein. The several serological reactions then follow as consequences of the properties of the sensitized surface and of the special environing conditions. The antibody is contained in the globulin fractions of immune serum, and appears to be a globulin with physico-chemical differences from normal serum globulin.

Antisera against several strains of acid-fast bacteria have been separated into their euglobulin, pseudoglobulin and albumin fractions. The globulin fractions have been found to possess the essential properties of bacteriotropic sera: thus they alter the bacterial surface properties, and, in quantitative correspondence, cause agglutination and phagocytosis; these several effects withstand washing of the sensitized bacteria; the effects are little if at all affected by inactivation of the antisera before fractionation; the combination of antibody and antigen is serologically specific. The conclusion is drawn that the contact of antigen with fresh homologous immune serum results in the deposit on the antigen surface of a substance or substances contained in the globulin fractions of the antiserum; as a consequence of this surface deposit leucocytes can spread upon and engulf the antigen.

1. The effect of HCl, NaOH, CO(2), and NH(3) on the volume of unfertilized Arbacia eggs was tested over a wide range of pH values. 2. No swelling occurred, except in HCl solutions, and there not until after injury or death had occurred. 3. Whereas the volume of erythrocytes and of proteins such as gelatin is known to be dependent on the pH of the solution, such a relation does not exist in the case of living and uninjured cells, at least of the type tested.

The work reported in this and in previous papers (1, 7) demonstrates the following relations for acid-fast bacteria and rabbit polymorphonudear leucocytes: 1. The combination of a substance or substances present in fresh immune rabbit serum, heated or unheated, or in fresh unheated normal rabbit serum, with a substance or substances in the bacterial surface causes an increase in cohesiveness, decrease in surface potential difference and characteristic alteration in wetting properties of the bacteria, and prepares the bacteria for phagocytosis. 2. (a) The effective substance or substances in the serum may become so altered as the result of heating or aging that combination with the bacterial surface, while causing changes in bacterial surface properties indistinguishable by the present physical-chemical tests from these just mentioned, may not lead to phagocytosis, or may lead to phagocytosis with a prezone not paralleled by a prezone in the changes in surface properties. (b) Sensitization of bacteria with human sera causes changes in surface properties similar to those caused by rabbit sera, but does not lead to phagocytosis by rabbit leucocytes. The spreading requirements of rabbit polymorphonuclear leucocytes are evidently highly selective.

Sensitization with increasing concentrations of homologous immune serum shifts the isoelectric point of the antigens studied progressively to the alkaline side. Antigens maximally sensitized with rabbit sera have shown isoelectric points of pH 5.6 to 5.8. The globulins precipitated or salted out of the same immune sera have been isoelectric at pH 5.1 to 5.2. The combination of antigen with antibody depends of course upon specific affinities; the surface properties of the sensitized antigen, agglutination and phagocytosis depend primarily upon the properties of the sensitizing serum substances combined with and deposited on the antigen surface.