COMPARATIVE STUDIES ON RESPIRATION : XXIV. THE EFFECTS OF CHLOROFORM ON THE RESPIRATION OF DEAD AND OF LIVING TISSUE.

1. Chloroform in low concentration (0.25 per cent) causes an increase in the rate of production of CO₂ in Ulva; this is followed by a decrease. In higher concentration (0.5 per cent) only a decrease is observed. 2. Assuming that the normal oxidation depends on the action of peroxide and peroxidase, experiments were made by placing Ulva in 1.0 per cent H₂O₂ and in Fe₂(SO₄)₃ (which acts like a peroxidase). The former diminishes the rate, the latter increases and subsequently decreases it. 3. When Ulva is killed in such a manner as to destroy the oxidizing enzymes, no CO₂ is produced unless H₂O₂ and Fe₂(SO₄)₃ are present. If to this mixture chloroform is added, the effect depends on the concentration of the iron. If the concentration is low there is an increase in the production of CO₂ followed by a decrease. If the concentration is high the rate appears to decrease from the start.     

STUDIES ON THE METABOLISM OF AUTOTROPHIC BACTERIA : II. THE NATURE OF THE CHEMOSYNTHETIC REACTION

In a study of chemosynthesis (the fixation of CO₂ by autotrophic bacteria in the dark) in Thiobacillus thiooxidans, the data obtained support the following conclusions: 1. CO₂ can be fixed by "resting cells" of Thiobacillus thiooxidans; the fixation is not "growth bound." 2. The physiological condition of the cell is of considerable importance in determining CO₂ fixation. 3. CO₂ fixation can occur in the absence of oxidizable sulfur in "young" cells. The extent of this fixation appears to be dependent upon the pCO₂. 4. CO₂ fixation can also occur under anaerobic conditions and the presence of sulfur does not influence such fixation. 5. However, in the CO₂ fixation by cells in the absence of sulfur, only a limited amount of CO₂ can be fixed. This amount is approximately 40 µl. CO₂ per 100 micrograms bacterial nitrogen. After a culture has utilized this amount of CO₂ it no longer has the ability to fix CO₂ but releases it during its respiration. 6. Relatively short periods of sulfur oxidation can restore the ability of cells to fix CO₂ under conditions where sulfur oxidation is prevented. 7. It is possible to oxidize sulfur in the absence of CO₂ and to store the energy thus formed within the cell. It is then possible to use this energy at a later time for the fixation of CO₂ in the entire absence of sulfur oxidation. 8. Cultures of Thiobacillus thiooxidans respiring on sulfur utilize CO₂ in a reaction which proceeds to a zero concentration of CO₂ in the atmosphere. 9. CO₂ may act as an oxidizing agent for sulfur. 10. Hydrogen is not utilized by the organism. 11. It is possible to selectively inhibit sulfur oxidation and CO₂ fixation.     

THE EFFECT OF CYSTINE AND GLYCOCOLL ON THE OXIDATION OF SODIUM LACTATE BY HYDROGEN PEROXIDE

The rate of production of CO₂ from sodium lactate when treated with H₂O₂ may be increased by the addition of a compound containing a sulfhydryl group, i.e. cystine. A small part of this increase in rate of CO₂ is due to the action of the amino group as shown by the action of glycocoll. The results tend to show that the mode of action of H₂O₂ is one of dehydrogenation and that the action of the cystine is comparable to the Atmungskörper of Meyerhof.     

THE EFFECT OF HYDROGEN ION CONCENTRATION ON THE PRODUCTION OF CARBON DIOXIDE BY BACILLUS BUTYRICUS AND BACILLUS SUBTILIS

1. The maximum rate of CO₂ production of Bacillus butyricus was found to be at a pH value of 7; of Bacillus subtilis at pH 6.8. If the pH value be raised or lowered there is a progressive decrease in the rate of production of CO₂. 2. Spontaneous recovery follows the addition of alkali to either organism, while addition of acid is followed by recovery only upon addition of an equivalent amount of alkali, and is not complete except when the amount of acid is very small.     

STUDIES ON THE METABOLISM OF THE AUTOTROPHIC BACTERIA : III. THE NATURE OF THE ENERGY STORAGE MATERIAL ACTIVE IN THE CHEMOSYNTHETIC PROCESS

In the autotrophic bacterium, Thiobacillus thiooxidans, the oxidation of sulfur is coupled to transfers of phosphate from the medium to the cells. CO₂ fixation is coupled to transfers of inorganic phosphate from the cells to the medium and is dependent, in the absence of concomitant sulfur oxidation, upon the amount of phosphate previously taken up during sulfur oxidation. The energy reservoir, which is formed by sulfur oxidation in the absence of CO₂ and which can be released for the fixation of CO₂ under conditions which do not permit sulfur oxidation, is a phosphorylated compound and the data suggest that the energy is stored in the cell as phosphate bond energy. It is possible to oxidize sulfur at a constant rate for hours in the absence of CO₂. The phosphate energy formed during this process is probably released by cell phosphotases. It is possible to inhibit these phosphotases by means of inorganic phosphate and thus to inhibit sulfur oxidation in the absence of CO₂. In the presence of CO₂, where alternative uses for the phosphate energy are available, the inhibition is relieved. Sulfur oxidation (energy input) is coupled, not to CO₂ fixation, but to phosphate esterification. CO₂ fixation (energy utilization) is coupled with phosphate release.     

COMPARATIVE STUDIES ON RESPIRATION : V. THE EFFECT OF ETHER ON THE PRODUCTION OF CARBON DIOXIDE BY ANIMALS.

1. The experiments on frog tadpoles show that with 0.15, 0.37, and 0.55 per cent ether solutions there is a decrease in CO₂ output. The effect is reversible. With these concentrations the breathing movements and body movements remained normal during the experiment. In 3.65 and 7.3 per cent ether there is a decrease of respiration followed by an increase which in turn is followed by a decrease. The increase may reach about three times the normal rate. The increase in the CO₂ output is accompanied by the peeling of the skin. The effect is irreversible. 2. Experiments on an aquatic insect, Dineutes assimilis Aube, show that in 7.3 per cent ether there is a decrease followed by an increase which in turn is followed by a decrease. There is no apparent disintegration of structures in the organism accompanying the increase. The effect is irreversible. 3. The experiments on frog eggs with 7.3 per cent ether show a result similar to that found in aquatic insects. 4. Experiments on Fundulus embryos show that with 0.73 per cent ether there is a reversible decrease in the rate of CO₂ production. In 3.65 per cent ether there is a temporary decrease followed by an increase, after which the rate begins to fall off. In 7.3 per cent ether there is an immediate increase amounting to 307 per cent which is followed by a decrease. The increase in the 3.65 and 7.3 per cent ether is accompanied by irreversible changes leading to death. The decrease found in 0.73 per cent ether is not sufficient to cause narcosis, as is shown by experiments on which the same decrease is produced by lowering the temperature. 5. These experiments show that narcosis is not due to asphyxia. The action of anesthetics is due to some other cause than the effect on respiration. There is a difference between the animals studied and the plants described in this series of articles, since in animals the increase in the CO₂ output is accompanied by irreversible changes leading to death, while this is not necessarily the case in plants. The reversible (narcotic) action of ether on the animals studied was accompanied by a decrease in the carbon dioxide output; in plants this is not ordinarily the case. These facts are of considerable interest, but their interpretation must be left to future investigation.     

THE INFLUENCE OF LIGHT AND CARBON DIOXIDE ON PHOTOSYNTHESIS

1. An optical system is described which furnishes an intensity of 282,000 meter candles at the bottom of a Warburg manometric vessel. With such a high intensity available it was possible to measure the rate of photosynthesis of single fronds of Cabomba caroliniana over a large range of intensities and CO₂ concentrations. 2. The data obtained are described with high precision by the equation KI = p/(p ² _max. – p ²)^½ where p is the rate of photosynthesis at light intensity I, K is a constant which locates the curve on the I axis, and p _max. is the asymptotic maximum rate of photosynthesis. With CO₂ concentration substituted for I, this equation describes the data of photosynthesis for Cabomba, as a function of CO₂ concentration. 3. The above equation also describes the data obtained by other investigators for photosynthesis as a function of intensity, and of CO₂ concentration where external diffusion rate is not the limiting factor. This shows that for different species of green plants there is a fundamental similarity in kinetic properties and therefore probably in chemical mechanism. 4. A derivation of the above equation can be made in terms of half-order photochemical and Blackman reactions, with intensity and CO₂ concentration entering as the first power, or if both sides of the equation are squared, the photochemical and Blackman reactions are first order and intensity and CO₂ enter as the square. The presence of fractional exponents or intensity as the square suggests a complex reaction mechanism involving more than one photochemical reaction. This is consistent with the requirement of 4 quanta for the reduction of a CO₂ molecule.     

THE ACTION OF INHIBITORY NERVES ON CARBON DIOXIDE PRODUCTION IN THE HEART GANGLION OF LIMULUS

It has been shown in this paper that stimulation of the inhibitory nerves of the neurogenic heart of Limulus, which correspond to the vagus nerves of the vertebrate heart, results in a marked diminution of CO₂ production in the heart ganglion, while stimulation of the ganglion, leading to increased activity of the heart, leads also to increased CO₂ production by the ganglion. This shows that inhibition of the automaticity of this ganglion by the action of its inhibitory nerves consists, not in a process of blocking, but in a diminution of those chemical reactions in the ganglion cells which give rise to the production of CO₂.     

THE EXCRETION OF CARBON DIOXIDE BY FROG NERVE

1. Quiescent sciatic nerve of the frog discharges CO₂ at the average rate of 0.00876 mg. CO₂ per gram of nerve per minute. 2. Sciatic nerve steeped one minute in boiling water discharges CO₂ at first at a low rate and after an hour and a half not at all. 3. Degenerated sciatic nerve discharges CO₂ at a slightly higher rate than normal living nerve does. 4. Connective tissue from the frog discharges CO₂ at an average rate of 0.0097 mg. per gram of tissue per minute. 5. Assuming that a nerve is composed of from one-half to one-quarter connective tissue the CO₂ output from its strictly nervous components is estimated to be at a rate of 0.008 mg. CO₂ per gram of nerve per minute. 6. Stimulated sciatic nerve increases the rate of its CO₂ output over quiescent nerve by about 14 per cent. When this number is corrected for strictly nervous tissue the rate is about 16 per cent. 7. The increased rate of CO₂ production noted on stimulation in normal sciatic nerves was not observed when they were boiled, blocked, or degenerated. It was also not observed with stimulated strands of connective tissue.     

THE KINETICS OF PENETRATION : IX. MODELS OF MATURE CELLS

To imitate cells which have ceased to grow we have made models in which artificial sap is separated from the external solution by a non-aqueous layer (representing the protoplasm). A stream of CO₂ is bubbled through the artificial sap to imitate its production by the living cell. Potassium passes from the external solution through the non-aqueous layer into the artificial sap and there reacts with CO₂ to form KHCO₃: its rate of entrance depends on the supply of CO₂. Hence the increase of volume depends on the supply of CO₂ (as is probably true of the living cell). By regulating the supply of CO₂ and the osmotic pressure we are able to keep the volume and composition of the artificial sap approximately constant while maintaining a higher concentration of potassium than in the external solution. In these respects the model resembles certain mature cells which have ceased to grow.     

DYNAMICS OF NERVE CELLS : II. THE TEMPERATURE COEFFICIENTS OF CARBON DIOXIDE PRODUCTION BY THE HEART GANGLION OF LIMULUS POLYPHEMUS.

1. It is possible to determine by the colorimetric method the rate of production of carbon dioxide by the cardiac ganglion of Limulus. 2. Carbon dioxide formation in the cardiac ganglion was found to run parallel to the rate of heart beat for different temperatures. 3. The conclusion seems justified that the rate of cardiac rhythm of Limulus depends upon a chemical reaction in the nerve cells of the cardiac ganglion and that this reaction is associated with the production of carbon dioxide since the rate of beat and the rate of CO₂ production are similarly affected by changes in temperature.     

THE PRODUCTION OF CARBON DIOXIDE BY NERVE

1. A modified Osterhout respiratory apparatus for the detection of CO₂ from nerve is described. 2. The lateral-line nerve from the dogfish discharges CO₂ at first with a gush for half an hour or so and then steadily at a lower rate for several hours. 3. Simple handling of the nerve does not increase the output of CO₂; cutting it revives gush. 4. The CO₂ produced by nerve is not escaping simply from a reservoir but is a true nervous metabolite. 5. The rate of discharge of CO₂ from a quiescent nerve varied from 0.0071 to 0.0128 mg. per gram of nerve per minute and averaged 0.0095 mg. 6. Stimulated nerve showed an increased rate of CO₂ production of 15.8 percent over that of quiescent nerve. 7. The results of these studies indicate that chemical change is a factor in nerve transmission.     

THE EFFECT OF pH ON THE DIVISION RATE OF THE COCCOLITHOPHORID CRICOSPHAERA ELONGATA1

The division rate of Cricosphaera elongata was measured as a function of pH in a medium buffered with the CO₂-bicarbonate-carbonate system. The optimum pH for cell division of the coccolithophorid was 7.8. A change of the partial pressure of CO₂ in the medium from 0.03 to 5% did not affect the division rate. Between pH 6.4 and 7.8 changes in the bicarbonate concentration from 0.1 to 6.0 mm and carbonate concentration from 0.007 to 0.1 mm did not affect the rate of division. At loiv experimental pH, C. elongata was nonmotile and grew in clumps; at higher pH values, it was motile and solitary. Coccoliths were not found covering C. elongata if calcite was soluble in the medium.     

THE INFLUENCE OF AMMONIUM SALTS ON CELL REACTION

1. It may be shown by means of cells of the flowers of a hybrid Rhododendron which contain a natural indicator, by means of starfish eggs stained with neutral red, and by means of an "artificial cell" in which living frog''s skin is employed that increased intracellular alkalinity may be brought about by solutions of a decidedly acid reaction which contain ammonium salts. 2. These results are analogous to those previously obtained with the CO₂-bicarbonate system, and depend on the facts: (a) that NH₄OH is sufficiently weak as a base to permit a certain degree of hydrolysis of its salts; and (b) that living cells are freely permeable to NH₄OH (or NH₃?) and not to mineral and many organic acids, and presumably not at least to the same extent to ammonium salts as such.     

EFFECT OF SODIUM SULFATE ON THE PHAGE-BACTERIUM REACTION

Bacteriophagy taking place in the presence of M/8 Na₂SO₄ has the following pronounced characteristics: A. Time of lysis is considerably prolonged. B. The bacteria take up less than the normal amount of phage. C. Phage production occurs at one-third the customary rate. D. It takes four times as much phage to lyse a Na₂SO₄-treated bacterium than a normal one. E. Bacterial growth is not affected by Na₂SO₄. The lag phase and the lowered rate of phage production can be attributed to the Na₂SO₄ effect on the cell surface. Less phage is taken up by the cells and contact of phage with the bacterium''s precursor-producing mechanism is impeded.     

THE MECHANISM OF ACTION OF β-BUNGAROTOXIN

—β-Bungarotoxin, a presynaptically-acting polypeptide neurotoxin, caused an efflux from synaptosomes of previously accumulated γ-aminobutyric acid and 2-deoxy-d -glucose. The toxin-induced efflux of γ-aminobutyric acid occurred by a Na⁺ -dependent process while that of 2-deoxyglucose was Na⁺ -independent. These effects were also produced by treating synaptosomes with low molecular weight compounds, including fatty acids, that inhibit oxidative phosphorylation. After incubation with β-bungarotoxin, synaptosomes exhibited increased production of ¹⁴CO₂ from [U-¹⁴C]glucose and decreased ATP levels. β-Bungarotoxin treatment of various subcellular membrane fractions caused the production of a factor that uncoupled oxidative phosphorylation when added to mitochondria. Mitochondria from toxin-treated brain tissue exhibited a limitation in the maximal rate of substrate utilization. We conclude that β-bungarotoxin acts by inhibiting oxidative phosphorylation in the mitochondria of nerve terminals. This inhibition accounts for the observed β-bungarotoxin effects on synaptosomes and at neuromuscular junctions. We suggest that the effects on energy metabolism result from a phospholipase A activity found to be associated with the toxin.     

CARBON DIOXIDE FROM THE NERVE CORD OF THE LOBSTER

1. The nerve cord of the lobster (Homarus americanus Milne-Edwards) is very delicate and can be used as a living preparation for only a few hours after its removal from the animal. 2. During the first hour or so after removal it discharges CO₂ at a steadily decreasing rate beginning at about 0.20 mg. CO₂ per gram of cord per minute and ending at about 0.07 mg. 3. This discharge exhibits a steady decrease in rate and is not divisible into a period of gush and a period of uniform outflow as with the lateral-line nerve of the dogfish. It terminates in a very few hours with the complete death of the cord. 4. Both handling and cutting the cord temporarily increase the rate of CO₂ output. 5. The stimulated cord discharges CO₂ at a rate about 26 per cent higher than that of the quiescent cord, an increase of about 1.6 times that of the increase observed in the lateral-line nerve of the dogfish under similar circumstances.     

THE INFLUENCE OF HYPOCAPNIA UPON INTRACELLULAR pH AND UPON SOME CARBOHYDRATE SUBSTRATES, AMINO ACIDS AND ORGANIC PHOSPHATES IN THE BRAIN

Abstract— In order to evaluate the influence of hypocapnia upon the energy metabolism of the brain, lightly anaesthetized rats were hyperventilated to arterial CO₂ tensions of 26, 15 and 10 mm Hg respectively, with subsequent measurements of intracellular pH and of tissue concentrations of carbohydrate substrates, amino acids and organic phosphates. At P_co1= 26 there was a moderate increase in the intracellular pH but when the P_co2 was reduced further to 10 mm Hg the intracellular pH returned to normal, or slightly subnormal, values. The reduction in P_Co2 was accompanied by increased cerebral cortical concentrations of lactate, pyruvate, citrate, α-ketoglutarate, malate and glutamate and by decreased aspartate concentrations. It is concluded that the accumulation of metabolic acids explains the normal value for intracellular pH at very low CO₂ tensions. Previous results obtained in man indicate that there is an increased anaerobic production of lactic acid in the brain in extreme hypocapnia. At comparable CO₂ tensions the present results showed a small fall in phosphocreatine and a small rise in ADP. However, since the ammonia concentrations were normal or decreased and since there was an increase in citrate, the results give no direct support to the hypothesis of an activation of phosphofructokinase. Since the cerebral venous P_o2 was reduced to 20 mm Hg at an arterial CO₂ tension of 10 mm Hg the accumulation of acids was probably secondary to tissue hypoxia. However, since there was no, or only a very small, increase in the calculated cytoplasmic NADH/NAD⁺ ratio, it appears less likely that acids accumulated due to lack of NAD⁺.     

EFFECT OF CARBON DIOXIDE ENRICHMENT ON DEVELOPMENT OF THE FIRST SIX MAINSTEM LEAVES IN SOYBEAN

Many conclusions concerning plant responses to CO₂ enrichment have been based on assumptions of increased leaf size derived from observations of average leaf area measured at some time well into the growth period. The objectives of this study were to study the effect of elevated CO₂ on 1) the timing of mainstem leaflet appearance, 2) the rate and duration of leaflet expansion, and 3) the final area of individual leaflets of soybeans (Glycine max [L.] Merr. cv. Bragg) grown from seed at 348, 502, and 645 μl 1^–1 CO₂ concentrations. Central leaflet areas from mainstem trifoliolates 1–6 were measured every two days from time of appearance to full expansion. Leaflets tended to appear earlier in elevated CO₂ treatments; leaflets 2 through 6 appeared an average of 0.4 days earlier in the 502 μl 1^–1 treatment and 1.2 days earlier in the 645 μl 1^–1 treatment than in the 349 μl 1^–1 treatment. Relative rates of expansion were different among leaflets in their response to elevated CO₂; expansion rates of leaflets 1 and 4 were significantly higher at the highest CO₂ concentration. However, final area of leaflets was not affected by CO₂, or (in leaflet 5 only) was slightly smaller at the highest CO₂ treatment. Apparently, higher expansion rates of leaflets 1 and 4 at high CO₂ were offset by a tendency for decreased duration of expansion. It appears that there are morphological constraints on final leaflet area in soybean seedlings which limit the effects of elevated CO₂ on the early development of mainstem leaf area.     

EFFECTS OF CARBON DIOXIDE ON THE GROWTH AND MORPHOGENESIS OF MARSILEA

Increased concentrations of CO₂ in air (1–50%) cause young plants of Marsilea vestita to exhibit many characteristics of the water form when they are grown on a solid substrate under sterile conditions. Thus these plants have longer internodes, shorter petioles, more rectangular-shaped epidermal cells and fewer stomata on the lower leaf epidermis than controls grown in 0.03% CO₂. Over a 2-week period, dry weight increase is considerably greater in 12.5% and 25% CO₂ than in 0.03% CO₂. Fifty percent CO₂ is inhibitory to growth. CO₂-enriched air has the same morphogenetic effect when supplied in the light or the dark. Possible explanations for this effect are discussed.