A KINETIC ANALYSIS OF THE ENDOGENOUS RESPIRATION OF BAKERS' YEAST

The process of endogenous respiration of two strains of bakers'' yeast, Saccharomyces cerevisiae, was examined kinetically. The rate of respiration with respect to time in a non-nutrient medium was found to exhibit two phases: (a) a period of constant rate of O₂ consumption and CO₂ production (R.Q. = 1) characteristic of cells with ample concentrations of stored material; (b) a first order decline in rate of respiration with respect to time, where the rate was proportional to the concentration of some substrate, S. (R.Q. = 1 throughout second phase.) The nature of this substrate was reexamined and the evidence summarized confirms the notion that it is a carbohydrate, probably glycogen. These phases of endogenous respiration were shown to depend upon the age of the culture and the amount of substrate available.     

THE EFFECT OF URETHANE ON THE CONSUMPTION OF OXYGEN AND THE RATE OF CELL DIVISION IN THE CILIATE TETRAHYMENA GELEII

1. The inhibition of oxygen consumption produced by a series of concentrations of ethyl carbamate has been measured in the protozoan Tetrahymena geleii. 2. The relation found between the narcotic concentration and its effect on respiration leads to the conclusion that urethane has two distinct modes of action in this cell. The respiratory data can be accurately predicted by assuming that the inhibitor acts on two independent parallel respiratory systems. 3. Complete suppression of cell division in this organism is brought about by approximately 0.1 M urethane. 4. Urethane concentrations up to 0.1 M affect primarily only one of the two postulated respiratory systems. The mechanism of the narcosis of cell division in this organism by urethane thus appears to be inhibition of this "activity" system.     

THE PRODUCTION AND INHIBITION OF ACTION CURRENTS BY ALCOHOL

Suitable concentrations of ethyl alcohol (1 to 1.5 M) applied to a spot on a cell of Nitella lower the P.D. enough to cause action currents. The alcohol then suppresses action currents arriving from other parts of the cell and acts as a block. After the alcohol is removed the normal P.D. and irritability return. Similar experiments on the sciatic nerve and skin of the frog produced only a negative result.     

THE ACTION OF THE PLANT GROWTH HORMONE

1. Sections of Avena coleoptiles are found to show a considerable elongation when suspended in solutions of growth substance. 2. This elongation does not take place in the absence of O₂ and is inhibited by KCN and phenylurethane. 3. The rate of respiration of sections of coleoptiles is increased by the addition of growth substance in concentrations which cause growth. High concentrations of growth substance inhibit growth and also respiration. 4. The increase in respiration is inhibited by KCN and phenylurethane in the concentrations which inhibit normal respiration. These concentrations are the same as those which inhibit growth. 5. From 2, 3, and 4, it seems possible that the increase in respiration caused by growth substance may be an essential part of its action in growth.     

THE METABOLIC SYSTEMS INVOLVED IN DISSIMILATION OF CARBOHYDRATE RESERVES IN BAKERS' YEAST

Evidence is presented showing that the dissimilation of carbohydrate reserves in two strains of bakers'' yeast, Saccharomyces cerevisiae, is a purely respiratory process. Endogenous respiration is KCN-labile. Our own experiments together with various accounts and data given in the literature show that the same "oxygen-transporting mechanism" functions in both endogenous and exogenous metabolism. However, the lack of sensitivity of the endogenous system of reactions to low concentrations of monoiodoacetic acid, the absence of anaerobic CO₂ production, and the absence of alcohol production, demonstrate that fermentation is not involved in the dissimilation of the carbohydrate reserves. Throughout the experiments the endogenous respiration behaved functionally as a unitary system of reactions. The O₂ consumption and CO₂ production were parallel at all times; i.e., the R. Q. was consistently 1. Monoiodoacetic acid and KCN in concentrations from 10^–5 to 10^–1 molar affected both O₂ uptake and CO₂ production to the same extent. The only agents known to alter the value of the R. Q. were those which disrupted the normal protoplasmic structure, viz. grinding the cells with sand, plasmolyzing them with toluol and hypertonic salt solutions, or pressing them in a hydraulic press. These agents brought about a vigorous anaerobic CO₂ production accompanied by an accumulation of alcohol in the medium. The unitary character of endogenous respiration is exhibited only when the normal structure of the cell is kept intact; apparently it depends upon the maintenance of a chambered (or compartmental) architecture of the cell.     

THE INFLUENCE OF ALCOHOL UPON THE GROWTH OF SEEDLINGS

In this paper it is shown that if the dry seeds of the cantaloupe (Cucumis melo) are soaked for 3 hours in solutions of ethyl alcohol of concentration ranging from 2 to 16 per cent by volume, and then germinated and grown in distilled water in the dark, the total growth attained is greater by amounts ranging from 9 to 35 per cent than is that made by seeds treated in every way identically except that they are initially soaked in distilled water instead of alcohol. It is shown that this result is not due simply to differences in osmotic pressure in the different alcohol solutions. It is probably due to a simple selective action of the alcohol which eliminates the constitutionally weak and defective seeds.     

Plasmodium knowlesi: glycolytic intermediate levels of enzyme activities of infected rhesus monkey erythrocytes

In vitro glycolytic enzyme activities and in vivo glycolytic intermediate concentrations were assayed in Plasmodium knowlesi-infected rhesus monkey erythrocytes and control erythrocytes. The enzyme activities of infected erythrocytes were greater than controls indicating that P. knowlesi had its own glycolytic system and that parasite glycolysis was the source of the increased rate of glucose consumption by infected erythrocytes. The P. knowlesi glycolytic enzymes phosphofructokinase and hexokinase were less sensitive to acid inhibition than uninfected red cells.P. knowlesi-infected monkey erythrocytes and Plasmodium berghei-infected mouse erythrocytes had similar in vivo glycolytic profiles and in vitro enzyme activity increases.     

The role of interspecies differences in the ratio of choline-containing phospholipid fractions of rodent erythrocytes in response of these cells to the effect of membranotropic compounds

In in vitro experiments, interspecies differences were revealed in the erythrocyte responses in varied rodent species—laboratory mice (Mus musculus L.), tundra voles (Microtus oeconomus Pall.) and bank voles (Myodes glareolus Pall.)—to the effect of chemical agents able to interact with membrane lipids and disrupt the membrane structure (detergent Triton X-100, oxidative stress inductor AAPH, antioxidant ionol or BHT, uranyl ion). It was hypothesized that these differences are due to physicochemical peculiarities of the erythrocyte membrane structure, specifically, the ratio of choline-containing fractions of phospholipids (phosphatidylcholine and sphingomyelin). The use of blood erythrocytes as an in vitro experimental model to study the mechanisms of toxicity as well as antioxidant and membrane-protective properties of compounds of different nature was shown to imply the choice of an adequate source of erythrocytes in view of considerable speciesdependent structural specificity of the lipid component of mammalian erythrocyte membranes.     

THE GROWTH AND RESPIRATION OF THE AVENA COLEOPTILE

1. Transport of the plant growth hormone into the Avena coleoptile as well as the action of the hormone on cell elongation in the coleoptile are shown to depend upon aerobic metabolism. 2. Crystalline auxine, in contrast with impure preparations, affects neither the magnitude nor the respiratory quotient of coleoptile respiration. 3. Increasing age of the coleoptile cell decreases its rate of elongation much more than its rate of respiration. HCN or phenylurethane on the other hand decrease the two processes to the same extent, in spite of the fact that only a small portion of the energy liberated by respiration can be used in the mechanical process of growth. 4. From 2 and 3 it is concluded that processes of a respiratory nature but of relatively small magnitude form one or more integral steps in the chain of reactions by which the plant growth hormone brings about cell elongation.     

COMPARATIVE STUDIES ON RESPIRATION : IX. THE EFFECTS OF ANTAGONISTIC SALTS ON THE RESPIRATION OF ASPERGILLUS NIGER.

1. In the presence of 0.05 per cent dextrose the respiration of Aspergillus niger is increased by NaCl in concentrations of 0.25 to 0.5M, and by 0.5M CaCl₂. 2. Stronger concentrations, as 2M NaCl and 1.25M CaCl₂, decrease the respiration. The decrease in the higher concentrations is probably an osmotic effect of these salts. 3. A mixture of 19 cc. of NaCl and 1 cc. of CaCl₂ (both 0.5M) showed antagonism, in that the respiration was normal, although each salt alone caused an increase. 4. Spores of Aspergillus niger did not germinate on 0.5M NaCl (plus 0.05 per cent dextrose) while they did on 0.5M CaCl₂ (plus 0.05 per cent dextrose) and on various mixtures of the two. This shows that a substance may have different effects on respiration from those which it has upon growth.     

RECENT PROGRESS IN THE STUDY OF EARLY EVENTS IN MAMMALIAN FERTILIZATION*

During the past 25 years, great advances have been made in understanding the physiology, morphology and biochemistry of fertilization in invertebrate animal species. In contrast to this situation, there is a paucity of knowledge pertaining to mammalian fertilization. Major areas in which information is lacking are the nature of changes undergone by spermatozoa in preparation for fertilization, and the mechanisms involved in sperm penetration of the egg investments. The present state of knowledge of these events is outlined, and the weaknesses of some current concepts are evaluated. Fertilization of mammalian eggs in vitro seems an attractive method for studying gamete interaction, but experience has shown that numerous problems are associated with this technique. As a result, the information on mammalian fertilization that has been derived from studies conducted in vitro has fallen considerably short of expectations; some factors contributing to this discrepancy are described. Recent findings concerning the regulation of sperm motility and fertilizing ability seem to have considerable significance for mammalian fertilization in vivo and in vitro. These findings have been utilized to refine existing procedures; fertilization of hamster eggs in vitro has now been accomplished in the presence of numbers of spermatozoa comparable to those believed to be present at the site and time of fertilization in vivo. It is anticipated that this improved technique, by more closely approximating the physiological situation, will substantially assist the derivation of useful information from in vitro fertilization studies.     

THE INFLUENCE OF SODIUM CHLORIDE AND TEMPERATURE ON THE ENDOGENOUS RESPIRATION OF B. CEREUS

Measurements were made of the rate of consumption of oxygen by suspensions of B. cereus, in sodium chloride solutions of concentration up to 1.8 M and over a range of pH from 6.0 to 7.5. It was found: 1. That the temperature coefficient was independent of the presence of sodium chloride in concentrations between 0.2 and 1.8 M, although the rate of respiration was lowered considerably under these conditions. 2. That in the presence of concentrations of sodium chloride less than 0.2 M, the rate of respiration was increased, and so was the temperature coefficient. 3. That small changes in the temperature coefficient occurred when the pH was changed. The temperature coefficient was higher the higher the rate of respiration. These data may be more readily interpreted by the hypothesis that the temperature coefficient is controlled by some master reaction, than by that which supposes that the temperature coefficient is determined by protoplasmic viscosity.     

THE CHLOROPHYLL-PROTEIN COMPOUND OF THE GREEN LEAF

1. Aqueous extracts of spinach and Aspidistra leaves yield highly opalescent preparations which are not in true solution. Such extracts differ markedly from colloidal chlorophyll in their spectrum and fluorescence. The differences between the green leaf pigment and chlorophyll in organic solvents are shown to be due to combination of chlorophyll with protein in the leaf. 2. The effect of some agents on extracts of the chlorophyll-protein compound has been investigated. Both strong acid and alkali modify the absorption spectrum, acid converting the compound to the phaeophytin derivative and alkali saponifying the esterified groups of chlorophyll. Even weakly acid solutions (pH 4.5) denature the protein. Heating denatures the protein and modifies the absorption spectrum and fluorescence as earlier described for the intact leaf. The protein is denatured by drying. Low concentrations of alcohol or acetone precipitate and denature the protein; higher concentrations cause dissociation liberating the pigments. 3. Detergents such as digitonin, bile salts, and sodium desoxycholate clarify the leaf extracts but denature the protein changing the spectrum and other properties. 4. Inhibiting agents of photosynthesis are without effect on the absorption spectrum of the chlorophyll-protein compound. 5. The red absorption band of chlorophyll possesses the same extinction value in organic solvents such as ether or petroleum ether, and in aqueous leaf extracts clarified by digitonin although the band positions are different. Using previously determined values of the extinction coefficients of purified chlorophylls a and b, the chlorophyll content of the leaf extracts may be estimated spectrophotometrically. 6. It was found that the average chlorophyll content of the purified chloroplasts was 7.86 per cent. The protein content was 46.5 per cent yielding an average value of 16.1 parts per 100 parts of protein. This corresponds to a chlorophyll content of three molecules of chlorophyll a and one of chlorophyll bfor the Svedberg unit of 17,500. It is suggested that this may represent a definite combining ratio of a and b in the protein molecule.     

COMPARATIVE STUDIES ON RESPIRATION : VIII. THE RESPIRATION OF BACILLUS SUBTILIS IN RELATION TO ANTAGONISM.

1. In relatively low concentrations of NaCl, KCl, and CaCl₂ the rate of respiration of Bacillus subtilis remains fairly constant for a period of several hours, while in the higher concentrations, there is a gradual decrease in the rate. 2. NaCl and KCl increase the rate of respiration of Bacillus subtilis somewhat at concentrations of 0.15 M and 0.2 M respectively; in sufficiently high concentrations they decrease the rate. CaCl₂ increases the rate of respiration of Bacillus subtilis at a concentration of 0.05 M and decreases the rate at somewhat higher concentrations. 3. The effects of salts upon respiration show a well marked antagonism between NaCl and CaCl₂, and between KCl and CaCl₂. The antagonism between NaCl and KCl is slight and the antagonism curve shows two maxima.     

Bongkrekic acid and the adenosinetriphosphate requirement of malaria parasites

The avian malaria parasite Plasmodium lophurae, when removed from its host erythrocytes into an appropriate medium, develops extracellularly in vitro. This development was inhibited by bongkrekic acid at concentrations down to 2 μg/ml. Adenosine triphosphate at high concentrations partly reversed the inhibition. Bongkrekic acid also inhibited intraerythrocytic development in vitro of the human malaria P. falciparum.     

The Trw Type IV Secretion System of Bartonella Mediates Host-Specific Adhesion to Erythrocytes

Bacterial pathogens typically infect only a limited range of hosts; however, the genetic mechanisms governing host-specificity are poorly understood. The α-proteobacterial genus Bartonella comprises 21 species that cause host-specific intraerythrocytic bacteremia as hallmark of infection in their respective mammalian reservoirs, including the human-specific pathogens Bartonella quintana and Bartonella bacilliformis that cause trench fever and Oroya fever, respectively. Here, we have identified bacterial factors that mediate host-specific erythrocyte colonization in the mammalian reservoirs. Using mouse-specific Bartonella birtlesii, human-specific Bartonella quintana, cat-specific Bartonella henselae and rat-specific Bartonella tribocorum, we established in vitro adhesion and invasion assays with isolated erythrocytes that fully reproduce the host-specificity of erythrocyte infection as observed in vivo. By signature-tagged mutagenesis of B. birtlesii and mutant selection in a mouse infection model we identified mutants impaired in establishing intraerythrocytic bacteremia. Among 45 abacteremic mutants, five failed to adhere to and invade mouse erythrocytes in vitro. The corresponding genes encode components of the type IV secretion system (T4SS) Trw, demonstrating that this virulence factor laterally acquired by the Bartonella lineage is directly involved in adherence to erythrocytes. Strikingly, ectopic expression of Trw of rat-specific B. tribocorum in cat-specific B. henselae or human-specific B. quintana expanded their host range for erythrocyte infection to rat, demonstrating that Trw mediates host-specific erythrocyte infection. A molecular evolutionary analysis of the trw locus further indicated that the variable, surface-located TrwL and TrwJ might represent the T4SS components that determine host-specificity of erythrocyte parasitism. In conclusion, we show that the laterally acquired Trw T4SS diversified in the Bartonella lineage to facilitate host-restricted adhesion to erythrocytes in a wide range of mammals.     

PERMEABILITY OF ERYTHROCYTES TO RADIOACTIVE POTASSIUM

The diffusion coefficients for the exchange of potassium across the membrane of erythrocytes of humans, rats, and rabbits have been determined by the use of artificially radioactive potassium, both into and out of the erythrocytes both in vitro and in vivo. The diffusion coefficients found in minutes^–1 were 0.2 to 0.25 x 10^–3 for human, 0.32 to 0.665 x 10^–3 for rabbits, and 1.0 x 10^–3 for rat erythrocytes. Rabbit erythrocytes appear to be more permeable in vivo. Reasons are advanced to explain the failure of earlier workers to demonstrate appreciable exchange of potassium in erythrocytes.     

DEVELOPMENT OF EYE COLORS IN DROSOPHILA: SOME PROPERTIES OF THE HORMONES CONCERNED

The substance inducing the production of pigment in the eyes of vermilion brown mutants of Drosophila melanogaster has been shown to be a relatively stable chemical entity possessing true hormone-like activity. A simple method for obtaining hormone solutions has been developed involving extraction of dried wild type Drosophila pupae with ethyl alcohol and water. A logarithmic proportionality has been found to exist between the amount of hormone and the induced eye color. This relationship provides a simple method for the quantitative determination of hormone concentration in given extracts. Larvae and pupae of D. melanogaster contain an intracellular enzyme which inactivates the hormone in the presence of molecular oxygen. The hormone is not oxidized under ordinary conditions with either molecular oxygen or hydrogen peroxide. The hormone has been found to be an amphoteric compound with both acidic and basic groups and with a molecular weight between 400 and 600. The solubility and precipitation reactions of the hormone suggest its amino acid-like nature. However, the instability to heat, acid, and alkali, and its rather restricted occurrence indicate a rather complex specific structure.     

Effects of manganese on potassium outflow from erythrocytes and on respiration of rat liver mitochondria

In this work, effects of manganese on respiration of rat liver mitochondria and the rate of K⁺ outflow from rat erythrocytes are studied in a broad range of concentrations. It is shown that manganese ions at low concentrations (1 × 10^–7–3 × 10^–5 М) inhibit K⁺ outflow from rat erythrocytes; this can be used to prevent their lysis. At high concentrations (1 × 10^–4–1 × 10^–3 M), manganese activates K⁺ outflow from the erythrocytes but inhibits the valinomycin-induced outflow of the ion from the erythrocytes. This fact is an indication of manganese influence on physicochemical properties of membranes. At low concentrations manganese does not affect parameters of respiration and oxidative phosphorylation of rat liver mitochondria, while at high concentrations it exerts acceleration of the mitochondrial respiration, i.e., uncouples respiration from phosphorilation and, hence, inhibits ATP synthesis.     

Entamoeba histolytica. I. Aerobic metabolism

The respiration of intact trophozoites harvested from axenic cultures of Entamoeba histolytica was studied with the polarographic technique utilizing the Clark oxygen electrode. A typical Q_o2 value for the freshly harvested amebae was 1 μatom oxygen/mg protein/hr.It was conclusively demonstrated that this parasite, a putative anaerobe, not only consumes oxygen when provided, but has a high affinity for the gas.Added glucose, galactose, and ethanol increased the respiratory rates, whereas other carbohydrates were without effect on the endogenous respiration. Intermediates of the tricarboxylic acid cycle, amino and fatty acids did not stimulate the respiration of E. histolytica.Inhibitors of the mammalian respiratory chain (cyanide, antimycin) as well as agents that inhibit enzymes catalyzing the tricarboxylic acid cycle (malonate, fluoropyruvate, fluoroacetate, fluorocitrate) had little effect on the endogenous or glucose-supported respiration. Alkylating agents (iodoacetamide, iodoacetate), cinnamate, and N-ethylymaleimide strongly inhibited the oxygen consumption of E. histolytica. The chemotherapeutic agents, Paromomycin, Emetine and Metronidazole, at concentrations that inhibit growth in vitro, did not restrict the respiration.Storage of the trophozoites at 4 C led to progressive deterioraion of the parasites and loss of endogenous and glucose-supported respiration. The deterioration was paralled by loss of SH-materials from the amebae. Likewise, sonication or lysis with detergents abolished both the endogenous respiration and response to glucose.Exogenous NADH or NADPH evoked only marginal increases in oxygen consumption of the freshly harvested amebae, but were effective respiratory substrates with stored or sonicated organisms. Addition of vitamin K₃ greatly enhanced the endogenous and glucose-supported respiration of the intact amebae, as well as enhancing the response of stored or sonicated amebae to reduced pyridine nucleotides.