ELECTROENDOSMOSIS THROUGH MAMMALIAN SEROUS MEMBRANES : I. THE HYDROGEN ION REVERSAL POINT WITH BUFFERS CONTAINING POLYVALENT ANIONS.

It is shown that when a mammalian serous membrane bathed in dilute buffer is traversed by an electric current, liquid is caused to stream through the membrane toward the cathode when the pH value of the buffer is on the alkaline side of a certain critical hydrogen ion concentration. Streaming is toward the anode on the acid side of the reversal point. Simple means for studying this electroendosmosis quantitatively are described. The mean values of the reversal points in all cases studied with the present buffers lie between pH = 4.3 and 5.3. The membranes studied have been the mesentery of the living and dead animal, and the parietal pericardium and pleura, post mortem. The membranes of dogs, cats, rabbits, and two human pericardia have been studied. All these membranes are essentially sheets of connective tissue, bearing blood vessels, lymphatics, and nerves, and in some instances fat cells, and lined on each surface by a single layer of pavement mesothelial cells. Intercellular fibers form the major bulk of the lean membranes. These are predominantly collagenous except in the pleural region used, in which elastin fibers are present in large proportion. By using buffers alternately more acid or more alkaline than the reversal point, the direction of liquid flow across any given membrane site could be reversed an indefinite number of times. The time interval required is only that taken by the requisite manipulation in changing buffers and making the runs. The mean values of the reversal points for the several membranes when bathed in hypotonic, unbalanced buffer and in isotonic, physiologically balanced buffers showed only small and inconstant differences. The fat and lean pericardia similarly showed small difference in the positions of the mean reversal points. The apparent reversal points for the mesenteries of living animals proved to be lower than those for the mesenteries post mortem. This low value in the animals with functioning circulation is interpreted as essentially due to admixture with the buffers of buffer salts from the blood. Clear differences between the reversal points of the membranes in the first compared with later hours or days post mortem were not detected, with the possible exception of a small shift toward the acid range of the fat pericardium reversal points estimated several days post mortem. The reversal points with cat membranes were somewhat lower (more acid) than those of the dog. The approximate mean reversal points found with the citrate-phosphate buffers used were as follows: For mesenteries of living animals pH = 4.4; for mesenteries, post mortem, pH = 4.8; for cat pleuræ, post mortem, pH = 4.3; for dog pleuræ, post mortem, pH = 5.0; for lean and fat pericardia, post mortem, pH = 5.1. The mean reversal point of the two human pericardia was about pH = 5.0. Reversal points determined with buffers containing only monovalent anions are somewhat higher (less acid), as will be shown later. The bearing of these data on the question of the chemical composition of the surfaces of the fat cells of the serous membranes is discussed. Briefly, it is believed that proteins are probably present in important amount in these cell surfaces

ELECTROENDOSMOSIS THROUGH MAMMALIAN SEROUS MEMBRANES : III. THE RELATION OF CURRENT STRENGTH AND SPECIFIC RESISTANCE TO RATE OF LIQUID TRANSPORT. TRANSPORT RATE WITH SERUM.

The rate of electroendosmotic flow through dog and cat pericardia is found to be proportional to the current strength. The plots of current strengths against volumes of liquid transported in unit time are, in the better experiments, straight lines passing through the origin; the slopes of the lines are characteristic of the several systems. Data on transport rate with buffers of different specific resistances showed the following phenomena: 1. Decrease of the observed transport rate to a minimum between σ values of 95 and 60 ohms. 2. Changes in the membrane markedly affecting transport rate, at conductivities and osmotic pressures close to those of the blood. 3. Polarization of the membrane during the passage of current. The mean rate found for electroendosmotic transport across dog and cat serous membranes bathed in serum has been 0.19 to 0.30 (average, 0.25) c.mm. per minute per milliampere. The best experiments with dog serum and the living mesenteries of dogs under ether gave a mean rate of 0.29 c.mm. per minute per milliampere. These data, together with data from other sources, are believed to indicate a probability approaching certainty that electroendosmotic effects are a factor in glandular secretion

ELECTROENDOSMOSIS THROUGH MAMMALIAN SEROUS MEMBRANES : II. COMPARISON OF HYDROGEN ION REVERSAL POINTS WITH ACETATE AND WITH CITRATE-PHOSPHATE BUFFERS.

The hydrogen ion reversal points of human, dog, and cat serous membranes have been determined with acetic acid-sodium acetate buffer mixtures, and are compared with the reversal points of the same membranes estimated with citrate-phosphate mixtures. The values with acetate buffer are about one-quarter of a pH unit higher (more alkaline) for fat membranes and almost one-half a pH unit higher for lean membranes. The acetate values are believed to correspond more closely to the true hydrogen ion reversal points. The reversal points are again found to be characteristic for membrane and species. No evidence of a postmortem shift in reversal point has been found. The charge of the membranes even in the living animal is capable of ready and repeated reversal

ON THE MECHANISM OF THE SERUM SENSITIZATION OF ACID-FAST BACTERIA

Serum sensitization of the acid-fast bacteria causes two definite and directly observable changes in the bacterial surface: 1. A change from a surface readily wet by oil to a surface more readily wet by aqueous salt solution than by oil. This change is observed by microscopic examination of the bacteria in a saline-oil interface; thus detected, the surface alteration is said to constitute a "positive interface reaction." 2. An increased cohesiveness of the sensitized bacteria. This may be detected either by centrifuging the bacteria and then shaking up the sediment (resuspension reaction), or by observation of the clumps in the saline-oil interface. The interface reaction is serologically specific and confirms the existence of qualitative differences among acid-fast bacteria. The interface reaction parallels the binding of agglutinins as detected by the resuspension reaction, but not agglutination as ordinarily tested for. The interface reaction is less sensitive,—i.e., gives lower titers—than the resuspension reaction in about the average ratio of 1:3. The interface reaction in most instances runs approximately parallel to the complement fixation reaction; under at least one set of conditions, however, the interface reaction is correlated with the binding of agglutinin but not with the complement fixation reaction. How much of the bacterial surface must be covered with agglutinin in order to produce agglutination varies greatly with the bacterial strain used. The bacterial surfaces are modified by treatment with fresh normal sera in a manner quantitatively less but qualitatively not observably different from the effects of immune sera. Heating normal human, sheep, goat, or rabbit sera for 30 minutes at 56°C. has usually diminished but not abolished their effect on the bacterial surface. Similar inactivation of guinea pig sera left them without detectable effect on the bacterial surface. The agglutination prezone is shown to be due to interference by excess colloidal material with the collisions of the bacteria prerequisite to clumping. The prezone maybe abolished by centrifugation and resuspension of the sediment. Antibodies may be partially dissociated from the sensitized bacteria by alkali, with return of the bacterial surface toward its normal, unsensitized condition. A carbohydrate yielding on hydrolysis a positive pentose test has been detected in the specific alcohol extracts of acid-fast bacteria studied by Furth and Aronson.27 The tentative suggestion is made that the alcohol-soluble antigens of acid-fast microorganisms may be conjugated lipins owing their specificity to carbohydrate haptenes. Protective antipneumococcus globulins after heat denaturation have shown behavior in the saline-tricaprylin interface indistinguishable from that of maximally sensitized acid-fast bacteria. This strengthens the evidence suggesting that sensitized bacteria are coated with denatured globulin

THE PROCESS OF PHAGOCYTOSIS : THE AGREEMENT BETWEEN DIRECT OBSERVATION AND DEDUCTIONS FROM THEORY

The phagocyte, then, is a complex system delicately responsive to internal and external influences. Interfacial tensions, and under certain conditions viscosity, are critical factors in determining the ingestion of particles with which the phagocyte has come into contact. Deductions from the formulation of these factors by Fenn and Ponder are in agreement with observation and with experimental analysis. However, other and still unformulated forces also enter into the behavior of these remarkable cells

THE SURFACE COMPOSITION OF THE TUBERCLE BACILLUS AND OTHER ACID-FAST BACTERIA

Acid-fast bacteria in the boundary surface between salt solution and a test oil (tricaprylin) are spontaneously wet and enveloped by the oil. This behavior contrasts with that of all other cells studied by the interfacial tension method. Four strains of human tubercle bacillus and an atypical bovine strain are an exception to the first statement above. These have possessed stability in the saline-oil interface; this stability is slight, however, and not comparable with that of non-acid-fast bacteria. Acid-fast bacteria subjected to prolonged extraction with alcohol show resistance to wetting by oil comparable to that of non-acid-fast bacteria. These "defatted" bacteria nevertheless retain their acid-fast staining properties. Acid fastness cannot then depend on the integrity of a surface membrane. Study of the cataphoresis of acid-fast bacteria by Freund has rendered the presence of protein in the surface highly probable. We are forced then to regard the surface of acid-fast bacteria as complex, containing at least lipoid and protein. Not improbably also carbohydrate is present

THE DEFORMABILITY AND THE WETTING PROPERTIES OF LEUCOCYTES AND ERYTHROCYTES

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

ON THE SURFACE COMPOSITION OF NORMAL AND SENSITIZED MAMMALIAN BLOOD CELLS

The interfacial tension method has been applied to the study of the surface composition of mammalian blood cells and to certain other particles. Unsensitized erythrocytes and stromata possess only a small margin of stability in the interface and pass readily into the oil phase. Specifically sensitized erythrocytes and stromata possess much greater stability in the interface and pass into the oil only with considerable mechanical aid; characteristic deformations of the erythrocyte surface or the interface or both often result. With special immune sera prepared by Landsteiner and van der Scheer the quantitative relations are such as to indicate that the increased polarity of the sensitized erythrocyte surface is due to combination of the red cell surface lipoids with hemolytic sensitizer. These results are corroborative of the conclusion of Landsteiner and van der Scheer that erythrocytes contain specific lipoid-soluble antigens. The tentative conclusion is reached that with these anti horse-erythrocyte sera at least the agglutinins combine predominantly with the protein of the red cell surfaces. Fresh human leucocytes are spread and disintegrated by the interfacial stresses. After heat injury over the condenser with substage lamp the leucocytes typically do not enter the boundary surface. They are pushed before the advancing interface and, if their further advance is obstructed, bend the interface backward to form peninsulas and vacuoles. This change after heating is in the opposite sense to that to be expected from denaturation of the proteins of the protoplasm. Fresh oxalated rabbit platelets pass very easily into the oils. After heating over the substage lamp these elements also become less oil-miscible. The interfacial tension relations of blood cells, bacteria, and several cell products are tabulated

THE MODIFICATION OF ANTIBODIES BY FORMALDEHYDE

Certain strains of bacteria which have only minimal zeta potentials over a wide range of pH, and upon which surface deposits can be formed, afford a favorable means of studying certain chemical and physical properties of the surface deposits. Films of specific antibody-globulin upon these bacteria possess basic groups which can combine with formaldehyde. Combination of these groups with HCHO under the conditions of the present experiments shifts the isoelectric point of the sensitizing film toward the acid side by about 0.6 to 0.8 pH unit, and reduces the agglutinating tendency of the sensitizing film. Antibodies may be formalinized before combination with antigen without marked change in their specific combining affinities. The properties of the sensitizing films are similar whether formol treatment occurs before or after the antigen-antibody combination. The nature of the basic groups has been discussed

A PARADOXICAL RELATION BETWEEN ZETA POTENTIAL AND SUSPENSION STABILITY IN S AND R VARIANTS OF INTESTINAL BACTERIA

The relation between electrokinetic potential and suspension stability of four strains of non-flagellate intestinal bacteria has been studied. The smooth forms have ζ-potentials which approximate zero over a wide range of pH and salt concentration, yet nevertheless form stable suspensions. The rough variants have ζ-potentials which vary with pH and electrolyte concentration in the familiar way. The rough forms have values of ζ-potential critical for their suspension stability