Julian M. Tobias

Membrane model: Association of inorganic cations with phospholipid monolayers

Abstract The present work is relevant to the problem of identifying the membrane molecule or molecules which may provide negatively charged groups for ion exchange. The monolayer approach has been used and the following experiments done: (i) association of cations with polar groups of purified phospholipids has been measured in terms of surface pressure-area diagrams, (ii) adsorption of Ca2+ to the monolayer and its dependence on pH has been measured to determine the relation between ionization of the monolayer polar groups and ion-exchange properties, and (iii) the displacement of Ca2+ from phosphatidylserine films by Na+, K+, or H+ at different degrees of monolayer packing has been measured to determine whether or not one ion is more effective than another in displacing Ca2+. The following findings suggest that phosphatidylserine and phosphatidylethanolamine may provide negatively charged groups for ion exchange at cell surfaces: 1. (a) Ca2+ in the hypophase reduces the surface pressure (condenses the monolayer) as compared with Na+ or K+. This reduction in surface pressure is not changed by addition to the hypophase of Na2+ or K+ up to 10 mM but is completely prevented by either Na+ or K+ at 100 mM. 2. (b) Uptake of Ca2+ by phosphatidylsernie and phosphatidylethanolamine monolayers measured by isotope technique shows that there is 1 Ca2+ adsorbed per phosphatidylserine and 0.35 Ca2+ per phosphatidylethanolamine molecule at pH7. 3. (c) Displacement of Ca2+ adsorbed to phosphatidylserine films by Na+, K+, or H+. This experiment shows that monovalent cations are in competition with Ca2+ for the negatively charged sites of phosphatidylserine monolayers. 4. (d) More Ca2+ is displaced from phosphatidylserine films by either Na+ or K+ at low surface pressure than at high surface pressure. This is interpreted in terms of peri-polar groups volume available for ion intrusion. 5. (e) Relation between ionization of the polar groups of phosphatidylserine and phosphatidylethanolamine and ion-exchange properties measured by studying the adsorption of Ca2+ to the monolayers and its groups of phosphatidylserine and phosphatidylethanolamine, and the carboxyl group of phosphatidylserine seem to be ionized.

Effect of anesthetics and convulsants on brain acetylcholine content.

Conclusions 1. Both free and total acetylcholine content of the whole rat brain are higher after nembutal or chloroform anesthesia (diffuse diminution of activity) than in the unanesthetized animal. The free acetylcholine change is more convincing after chloroform, whereas the total acetylcholine change is more convincing after nembutal. The total acetylcholine content of the frog brain is similarly elevated after nembutal. 2. Neither free nor total acetylcholine content of rat brain differed significantly after the onset of strychnine or picrotoxin convulsions (diffuse increase of activity) from that found in quiet animals awake. Nor did strychnine alter total acetylcholine content of the frog brain. 3. With the methods used, normal whole rat brain has been found to contain about 0.7 μg of free and 2.9 μg of total acetylcholine per gram wet weight; frog brain about 4.9 μg of total acetylcholine per gram.

The Excitable System in the Cell Surface

A membrane model, composed of phospholipid and cholesterol, is described. The electrical resistance and hydration of this model can be controlled by manipulation of ambient ions and by current in ways strongly reminiscent of the behavior of living cells. The behavior of the model may resemble that of the membrane component of the cell. In addition, an interdependent, lipid-protein molecular structure may exist at the cell surface.

Nerve Conduction After Inactivation of Choline Esterase.

Conclusions Frog nerve, exposed to DFP in a concentration and for a time sufficient fully to inactivate its choline esterase, can still conduct impulses with entirely normal action potentials. Ch.E. is not essential to nerve conduction.

Measured Dose of Gamma Hexachlorocyclohexane (γ 666) Required to Kill Flies and Cockroaches, and a Comparison with DDT

Summary 1. The approximate LD50 for y 666 in the cockroach (Periplaneta americana) is 4.6 mg per kg when applied to the body surface and 3.4 mg per kg when injected intra-abdominally. These values are not significantly different. 2. An approximate equivalence, similar to that noted above. between surface and injection toxicity for the roach has been reported for DDT. This finding emphasizes the importance of the absorptive capacity of the insect body surface for contact poisons in contributing to the effectiveness of insecticides. 3. The approximate LD50 for y 666 applied to the body surface is 0.4 mg per kg for the newly emerged fly (Musca domestira). For the older adult the LD50 is about 0.8 mg per kg for Musca domestica and 0.6 mg per kg for Calliphora spp. This decrease in sensitivity as the fly ages is leas than that reported for DDT. 4. Gamma 666 is about twice as toxic as DDT for the cockroach (Periplaneta americana), and is distinctly more toxic than DDT for Musca domestica (newly emerged and adult) and Calliphora spp. (adult). 5. Both death and knockdown occur more rapidly after surface application of y 666 to Musca domestica and Periplaneta americana than after DDT.

The Effect of Exercise on Mortality of Animals Poisoned with Diphosgene

Conclusions It is clear that the worst that can be said of swimming shortly after diphosgene gassing at mid- or somewhat above mid-lethal doses is that it had no influence on final mortality among mice and rats or on survival time in dogs. There was, in fact, some indication, especially from the finding of a significant difference between survival of moderate early and late exercised mice, that moderate early swimming was beneficial or late swimming deleterious or both. It is striking that in all of the pooled data (see tables), and very often in individual experiments, differences noted, though not statistically significant, are consistently in the direction of increased survival for moderate early exercise in all 3 species, and in the direction of increased mortality for moderate or heavy late exercise in mice and heavy early exercise in dogs. The dog survival times and lung weights suggest that “moderate” and “heavy” exercise as distinguished in these experiments are physiologically as well as quantitatively, of different orders of magnitude. Similarly, in the mouse experiments there is no indication of any effect, beneficial or deleterious, of heavy early swimming, as opposed to the indicated beneficial influence of moderate exercise. Swimming seems a justifiable test of the effects of more natural types of exertion. The control experiments on mice show that the indifferent role of swimming or the perhaps beneficial effect of moderate early swimming does not represent an overcompensation of harmful exercise by a protective action of immersion in tepid water. If anything, swimming, as already indicated, may be responsible for the apparent ill effect of heavy “exercise” in dogs judged by the significantly greater pulmonary edema of heavy swimmers as opposed to controls. Finally, it may be pointed out that exercise by a diphosgene patient or other pulmonary irritant patient during the clinical latent period need not be expected a priori to be adding insult to injury. The water and blood demands of muscle during exercise could tend to delay the onset of edema. Once edema and attendant anoxaemia set in, however, increased oxygen demand becomes an undesirable feature of exercise. Since the time of onset of edema is variable, depending mainly on severity of exposure, which is in turn a function of more than just gas concentration and exposure duration, no particular time can be set as the limit of a safe period.

Membrane Interferometer Manometer

A sensitive indicator of pressure change has been constructed which utilizes a thin organic film as one of a pair of interferometer reflectors. Displacement of the film with respect to a fixed glass plate results in a shift of interference fringes. The film, being very thin, is extremely sensitive to pressure changes.

An Improved Capillary Microrespirometer

Instruments for the measurement of tissue and cell respiration have steadily moved, since the introduction of Warburgs convenient manometer, in the direction of smaller volume and greater sensitivity. Gerard and Hartline 1 took advantage of the greater stability afforded by reducing the tissue chamber of a volumeter to capillary dimensions (0.5 to 1.2 mm diameter) and having this inside the relatively large “differential” chamber. Index drop movements, followed with an ocular micrometer, were consistent over 5-minute intervals, even when corresponding to volume changes of about 0.01 cmm. The diver technique, introduced by Linderström-Lang, 2 , 3 is of the same order of sensitivity and consistency; and the electrical method 4 gives promise of superior performance. We have further developed the capillary method so that it is convenient to follow the respiration of 10 tissue samples at once, and it is possible to measure absolute gas volume changes of 0.001 cmm, minute by minute, with an error of some 8% minute by minute, under 1% for longer intervals. The respiration even of bits of frog sciatic nerve weighing a fraction of a milligram can thus be followed at half-minute intervals. The tissue chamber, also containing filter paper bits soaked with acid or alkali and separated from each other and the tissue by dry paper guards, is a short length of capillary of 1.2-1.5 mm internal diameter. The “open” end of this, after the insertion of materials, is plugged with plasticine. Into the other end has been cemented a fine drawn capillary of about 0.2 mm diameter. At the close of the experiment, this capillary is broken off and its end diameters accurately measured by end on examination with an ocular micrometer.

Syringe Oxygen Cathode for Measurement of Oxygen Tension in Solution and in Respiratory Gases

A method is described for measuring oxygen tension in Ringer solution which utilizes the principle of the polarograph, but substitutes a platinum wire cathode for the usual dropping mercury electrode. The wire cathode and a calomel anode are mounted in the piston of a hypodermic syringe into which the solution is aspirated for analysis. Sensitivity and reproducibility are evaluated. Extension of the method to the analysis of gaseous oxygen is also described.