The course of respiration during the life cycle of Chlorella cells

Endogenous and glucose respiration were studied during the life history of Chlorella pyrenoidosa. A generalized picture of the course of respiration during the life cycle is suggested. At the liberation of daughter cells from the wall of the mother cell, or soon after, the respiration rate reaches its lowest level. If the daughter cells are placed in light the respiration rate rapidly increases with time, soon reaches a maximum, and then declines slowly. Two factors are important in the initial increase—the early developmental stage of the cells and the influence of light. In autotrophically developing algae the parts played by developmental processes and by light have not been separated. Direct activation of respiratory enzymes by light, in addition to the level of respiratory substrate, cannot be excluded. The decline of respiration rate over most of the cell history seems to have no connection with light and is probably bound to the developmental processes per se. Darkening the suspension interrupts growth and induces liberation of daughter cells, with concomitant faster decrease in respiration rate. The rate of respiration of small daughter cells decreases in darkness only slowly with time. Illumination seems necessary to bring these cells back to a high level of respiratory activity.     

Effect of amphotericin B on the enzyme system of Candida albicans]

The mechanism of amphotericin B action was studied with the aid of cytochemical methods providing determination of the activity of the 4 main enzymes characterizing the cell energetics, i. e. succinate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase and glucose-6-phosphate dehydrogenase inside the cell. With an increase in the concentration of amphotericin B in the medium the activity of all the 4 enzymes decreased, the percentage of the inactive cells being regularly increased. Changes in the fermentative activity of C. albicans as dependent on the incubation time with the antibiotic were studied. Only the respiration activity decreased in 2 hours. As a result of a 4-hour exposure to the polyen in the cells of C. albicans the activity of the lactic acid fermentation, respiration through succinate dehydrogenase and activity of the pentose shunt decreased 1.5--2 times. In 24 hours of incubation the activity of the above decreased 80--90 per cent as compared to the activity of the initial culture.     

Respiration of bloodstream forms of the parasite Trypanosoma brucei brucei is dependent on a plant-like alternative oxidase

CoQ links the sn-glycerol-3-phosphate dehydrogenase and oxidase components of the cyanide-insensitive, non-cytochrome-mediated respiratory system of bloodstream African trypanosomes. In this and other characteristics, their respiratory system is similar to the alternative oxidase of plants. The parasites contain 206 ng of CoQ9 mg protein-1 which co-sediments with respiratory activity. The redox state of this CoQ responds in a manner consistent with respiratory function: 60% being in the reduced form when substrate is available and the oxidase is blocked; 13% being in the reduced form when the oxidase is functioning and there is no substrate. The addition of CoQ to aceton-extracted cells stimulates salicylhydroxamic acid-sensitive respiration by 56%. After inhibition of respiration by digitonin-mediated dispersal of the electron transport components, liposomes restore 40% of respiratory activity while liposomes containing CoQ restore 66% of this activity. A less hydrophobic analogue, reduced decyl CoQ, serves as a direct substrate for the trypanosome oxidase supporting full salicylhydroxamic acid-sensitive respiration. After digitonin disruption of electron transport, the nonreduced form of this synthetic substrate can reestablish the chain by accepting electrons from dispersed sn-glycerol-3-phosphate dehydrogenase and transferring them to the dispersed oxidase. Similarities between the alternative oxidase of plants and the oxidase of the trypanosome respiratory system include: mitochondrial location, lack of oxidative phosphorylation, linkage of a dehydrogenase and an oxidase by CoQ, lack of sensitivity to a range of mitochondrial inhibitors, and sensitivity to a spectrum of inhibitors which selectively block transfer of electrons from reduced CoQ to the terminal oxidase but do not block electron transfer to the cytochrome bc1 complex of the mammalian cytochrome chain.     

Effect of cultivation conditions on the activity of respiration of Saccharomyces bayanus]

The effect of sugar and ammonium nitrogen content in the initial nutrient medium as well as the cultivation temperature on the respiration activity and respiratory quotient of yeast used in the champagne manufacture was studied. An addition of ammonium nitrogen stimulated respiratory processes in the yeast cell. The respiration activity increased with an increase of pH to 4.0. With an elevation of the sugar content of the substrate the respiratory quotient increased resulting in an uneconomic utilization of sugar. The maximum respiration activity and minimum respiratory quotient occurred at a temperature of 20 degrees.     

Mitochondrial respiration defects modulate differentiation but not proliferation of hematopoietic stem and progenitor cells

Mitochondrial energy production is involved in various cellular processes. Here we show that ATP content is significantly increased in lineage-restricted progenitor cells compared with hematopoietic stem and progenitor cells (HSPCs) or more differentiated cells. Transplantation analysis using a mouse model of mitochondrial disease revealed that mitochondrial respiration defects resulted in a significant decrease in the total number and repopulating activity of bone marrow cells, although the number of HSPCs increased. The proliferative activity of HSPCs and lineage-restricted progenitor cells was not impaired by reduction of ATP content and there seems to be no associated increase in reactive oxygen species levels and apoptosis. Our findings indicate that mitochondrial respiration defects modulate HSPC commitment/differentiation into lineage-restricted progenitor cells.     

Functional characteristics of light and dark cells]

Comparison of peculiarties attending the structure of mitochondria in the dark and light cells with the results of studying the respiration of condensed, intermediate and orthodoxic mitochondria showed that dark cells containing condensed mitochondria were at the state of rest or specific activity associated with a high level of bioenergetic and biosynthetic processes. Light cells with a preponderant content of intermediate mitochondria are characterized by the state of active specific activity accompanied by a high energy potential, intensified energy expenditure and a reduction of the level of biosynthetic processes. Light cells with a prevalence of orthodoxic mitochondria are characterized by a low level of energy provision and a predominance of the processes of decomposition of structures over their resynthesis.     

Contribution of the Phosphorylable Complex I in the Growth Phase-Dependent Respiration of C6 Glioma Cells in Vitro

The energy metabolism of rat C6 glioma cells was investigated as a function of the growth phases. Three-dimensional cultures of C6 cells exhibited diminished respiration and respiratory capacity during the early growth phase, before reaching confluence. This decrease in respiration was neither due to changes in the respiratory complex content nor in the mitochondrial mass per se. Nevertheless, a quantitative correlation was found between cellular respiration and the rotenone-sensitive NADH ubiquinone oxidoreductase (i.e. complex I) activity. Immunoblot analysis showed that phosphorylation of the 18 kDa-subunit of this complex was associated with the growth-phase dependent modulation of complex I and respiratory activity in C6 cells. In addition, by using forskolin or dibutyryl cAMP, short-term activation of protein kinases A of C6 cells correlated with increased phosphorylation of the 18-kDa subunit of complex I, activated NADH ubiquinone oxidoreductase activity and stimulated cellular respiration. These findings suggest that complex I of C6 glioma cells is a key regulating step that modulates the oxidative phosphorylation capacity during growth phase transitions.     

Inhibition of Krebs cycle and activation of glyoxylate cycle in the course of chronological aging of Saccharomyces cerevisiae. Compensatory role of succinate oxidation

We investigated oxidative processes in mitochondria of Saccharomyces cerevisiae grown on ethanol in the course of chronological aging. We elaborated a model of chronological aging that avoids the influence of exhaustion of medium, as well as the accumulation of toxic metabolites during aging. A decrease in total respiration of cells and, even more, of the contribution of respiration coupled with ATP-synthesis was observed during aging. Aging is also related with the decrease of the contribution of malonate-insensitive respiration. Activities of citrate-synthase (CS), alpha-ketoglutarate dehydrogenase (KGDH) and malate dehydrogenase (MDH) were threefold decreased. The activity of NADP-dependent isocitrate dehydrogenase (NADP-ICDH) decreased more significantly, while the activity of NAD-dependent isocitrate dehydrogenase (NAD-ICDH) fell even greater, being completely inactivated on the third week of aging. In contrast, succinate dehydrogenase (SDH), enzymes of glyoxylate cycle (GCL) (isocitrate lyase (ICL) and malate synthase (MLS)), and enzymes of ethanol oxidation (alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ACDH)), were activated by 50% or more. The behavior of oxidative enzymes and metabolic pathways are apparently inherent to a more viable, long-lived cells in population, selected in the course of chronological aging. This selection allows cells to reveal the mechanism of their higher viability as caused by shunting of complete Krebs cycle by glyoxylate cycle, with a concomitant increased rate of the most efficient energy source, namely succinate formation and oxidation. Thiobarbituric-reactive species (TAR species) increased during aging. We supposed that to be the immediate cause of damage of a part of yeast population. These data show that a greater succinate contribution to respiration in more active cells is a general property of yeast and animal tissues.     

Effect of cycloheximide on the ultrastructure and several metabolic processes of the cells of an SPEV culture. II

The action of cyclohexamide (in doses of 1 and 10 mkg/ml in the course of 24 hours) on porcine embryo kidney cells in culture is accompanied by a powerful suppression of protein and DNA synthesis, mitotic activity, a weaker suppression of RNA synthesis, a lowering of the activity of succinate-, lactate- and alpha-glycerophosphate-dehydrogenase, which leads to disorders in the ultrastructure of the cells. After incubation of cells in a fresh medium (in the course of 18, 24 hours) there occurs a total restoration of the ultrastructure of the nuclei, granular endoplasmatic reticulum, mitochondria, due to the repairing and strong intensification of synthetic processes, respiration and glycolysis, mitotic activity of the cells.     

Effect of magnetic field on the process of cell respiration in mitochondria of rats

Exposure of rats to static magnetic field 1 hour daily for a period of 7 weeks (7 days a week) leading to disturbances of the respiration processes in the mitochondria of liver cells. The rate of respiration through NADH dehydrogenase, succinic dehydrogenase and cytochrome oxidase was dependent on both the duration and the intensity value of the field applied. The animals showed greater sensitivity to the action of a 0.008 T magnetic induction field than to that of 0.15 T. The observed changes were reversible after 3 months since the everyday exposure had been stopped.     

Control of brain slice respiration by (Na+ + K+)-activated adenosine triphosphate and the effects of enzyme inhibitors.

The involvement of membrane (Na+ + K+)-ATPase (Mg2+-dependent, (Na+ + K+)-activated ATP phosphohydrolase, E.C. 3.6.1.3) in the oxygen consumption of rat brain cortical slices was studied in order to determine whether (Na+ + K+)-ATPase activity in intact cells can be estimated from oxygen consumption. The stimulation of brain slice respiration with K+ required the simultaneous presence of Na+. Ouabain, a specific inhibitor of (Na+ + K+)-ATPase, significantly inhibited the (Na+ + K+)-stimulation of respiration. These observations suggest that the (Na+ + K+)-stimulation of brain slice respiration is related to ADP production as a result of (Na+ + K+)-ATPase activity. However, ouabain also inhibited non-K+ -stimulated respiration. Additionally, ouabain markedly reduced the stimulation of respiration by 2,4-dinitrophenol in a high (Na+ + K+)-medium. Thus, ouabain depresses brain slice respiration by reducing the availability of ADP through (Na+ + K+)-ATPase inhibition and acts additionally by increasing the intracellular Na+ concentration. These studies indicate that the use of ouabain results in an over-estimation of the respiration related to (Na+ + K+)-ATPase activity. This fraction of the respiration can be estimated more precisely from the difference between slice respiration in high Na+ and K+ media and that in choline, K+ media. Studies were performed with two (Na+ + K+)-ATPase inhibitors to determine whether administration of these agents to intact rats would produce changes in brain respiration and (Na+ + K+)-ATPase activity. The intraperitoneal injection of digitoxin in rats caused an inhibition of brain (Na+ + K+)-ATPase and related respiration, but chlorpromazine failed to alter either (Na+ + K+)-ATPase activity or related respiration.     

土壤微生物总活性研究方法进展

微生物总活性是指在某一时段内微生物所有生命活动的总和,它直接决定着微生物行使生理、生态功能的能力,是微生物学研究的热点,也是难点。迄今为止,还没有建立直接测定微生物总活性的方法,只能用一些相关指标来间接反映它。目前常用的指标主要包括微生物的呼吸速率、生长速率以及胞内RNA含量等。与其它一些基质和环境相比,测定土壤中的微生物总活性更为困难。通过总结研究土壤微生物总活性常用的3种方法,在简略概括传统的土壤微生物呼吸测定法的基础上,详细介绍了放射性同位素标记法和RNA直接表征法的原理和操作流程,整理归纳了一些重要应用案例,比较分析了不同方法的优缺点,以期为选择研究土壤微生物总活性的适宜方法提供依据。     

Stimulation by thyroid hormone of coupled respiration and of respiration apparently not coupled to the synthesis of ATP in rat hepatocytes.

Maximal rates of O2 uptake by hepatocytes from hypothyroid, euthyroid, and hyperthyroid rats, in the absence of uncoupling agents or ionophores, were achieved by simultaneously stimulating ureogenesis and gluconeogenesis. Rates were increased by pretreatment of the donor animals with thyroid hormone. Only a minor part of the increase could be attributed to stimulation of the activity of plasma membrane (Na+ + K+)-ATPase. No synthesis of glycogen occurred, nor was there any evidence of pyruvate cycling under the conditions used, thus ruling out these processes as potential sources of ATP turnover. Calculation of the O2 uptake necessary to satisfy the energy requirements of gluconeogenesis and ureogenesis, based on anticipated ATP demand and an assumed P:O ratio of 3:1, invariably yielded a theoretical quantity that was less than the experimentally measured increase in O2 uptake. The difference between measured and calculated rates of respiration, possibly representing noncoupled respiration, was related to the thyroid status of the animal, being greatest in cells from hyperthyroid rats. In another experimental approach the O2 uptake associated with ATP synthesis was inhibited by the addition of oligomycin to hepatocytes incubated in the presence of substrates, thus abolishing ATP-coupled respiration. The magnitude of the ATP-coupled as well as the residual respiration was increased in response to thyroid hormone. Thyroid hormone increased the turnover of intramitochondrial ATP. These results imply that a considerable portion of the thermogenic effect of thyroid hormone may be mediated by a stimulatory action on metabolism not directly associated with extramitochondrial ATP-dependent synthetic processes.     

Influence of glutamic acid on the endogenous respiration of Bacillus subtilis

Clifton, C. E. (Stanford University, Stanford, Calif.), and John Cherry. Influence of glutamic acid on the endogenous respiration of Bacillus subtilis. J. Bacteriol. 91:546-550. 1966.-Amino acids serve as the major initial endogenous substrate for Bacillus subtilis. The endogenous activity of freshly harvested washed cells is high and falls off rapidly with time of shaking at 30 C to lower but still significant levels. The rate of O(2) consumption after the addition of glutamic acid also decreases as the cells age, but more slowly than noted for endogenous respiration. When cells were fed glutamate as soon as possible after harvesting, an apparent stimulation of endogenous respiration was noted. However, endogenous activity was inhibited if the cell suspensions were shaken for at least 1 hr before addition of the glutamate. Similar results were obtained with glycerol or glucose as exogenous substrates. Variation in rates of respiration with age of the cells, inherent instability of B. subtilis, and possible utilization of substances initially excreted by the cells appear to account for the variations noted regarding the influence of an exogenous substrate on endogenous respiration.     

Respiration rates, ATP content and ionic regulation in hepatocytes of starving lamprey during the pre-spawning period of their life cycle

During the winter pre-spawning migration, lampreys Lampetra fluviatilis stop feeding, and their liver metabolism is reduced substantially. Aerobic ATP production in hepatocytes decreased to one third and ATP content decreased by 50% as compared with the values in autumn. In spite of the decrease of endogenous and phosphorylating (oligomycin-sensitive) respiration in winter, the oxygen consumption used to drive sodium and potassium pumping through Na,K-ATPase activity (ouabain-sensitive respiration) remained virtually constant. Consequently its share in phosphorylating respiration increased from 16·3% in November to 54·2% in February. Potassium influx was similar within the range of ATP content between 2·5 and 1 nmol 10⁻⁶ cells and decreases only in hepatocytes which contained <0·8 nmol ATP 10⁻⁶ cells.     

Effect of disinfectants on the metabolism of<Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar enteritidis

Antimicrobial activity of 19 commercially manufactured disinfectant substances on a Salmonella enteritidis strain was determined. The substances represented 8 quaternary ammonium salts (QAS) and 10 QAS combined with other additives. The antimicrobial efficacy was characterized by influencing the growth of bacterial cells expressed as MIC and ED50 values as well as by the inhibition of the incorporation rate of 14C-adenine and 14C-leucine. According to their efficacy the disinfectants were divided into three groups: (1) substances with strong inhibitory effect (MIC 6-45 micrograms/L) such as Diesin forte, Hexaquart plus, Neoquat S, Triquart, Almyrol, Hexaquart S, ID212, ID213 and Microbac forte; (2) substances with good antibacterial efficacy (MIC 90-780 micrograms/L); (3) substances with MIC values > 780 micrograms/L (up to 3120 micrograms/L). Cetrimide had very low activity (MIC 3.12-6.25 mg/L). The effect of disinfectants on the biosynthetic processes was expressed by R values (IC50(Ade):IC50(Leu)); all these values were < 1 except Benzalkonium chloride, FD312, Divoquat forte, 5P plus, Almyrol, Hexaquart S and Hexaquart plus. Low R values suggested interference of these substances with the synthesis of both nucleic acids and proteins. All substances except 5P plus caused an inhibition of endogenous respiration. The most effective were Almyrol, Diesin forte, Microbac forte and Neoquat which completely inhibited respiration at 190 mg/L. Kvart showed the lowest effect on the respiration over the whole concentration range. The disinfectants also suppressed growth of S. enteritidis, probably by interfering with energy-yielding and-requiring processes in the cells.     

Poliovirus induces an early impairment of mitochondrial function by inhibiting succinate dehydrogenase activity

Poliovirus infection of COS-1 and T47D cells caused a rapid decrease in total cell respiration, and this was attributed to an inhibition of mitochondrial respiration. The stimulation of mitochondrial respiration by pyruvate plus malate or succinate was impaired in saponin-permeabilised cells. However, this inhibition could be overcome by the addition of N,N,N',N'-tetramethyl-1, 4-phenylenediamine and ascorbate. The activity of succinate dehydrogenase was impaired in parallel with the inhibition of mitochondrial respiration during poliovirus infection. This shows that mitochondrial function is profoundly altered during poliovirus infection and that this occurs primarily through inhibition of electron flow at complex II of the mitochondrial respiratory chain.     

Phosphorylant capacity study and lactate mitochondrial oxidation in frozen bovine sperm

Frozen-stored bovine sperm-pellets of proven fertility were used, and the response to respiratory chain effectors was studied, thus demonstrating the energy conservation capacity. It was further observed that the assayed suspensions used lactate oxidatively, which proves the LDH-X mitochondrial activity (the presence of oxidative substrates is fundamental in capacitation and acrosome reaction processes). The suspensions were treated with 10mM phosphate buffer hypotonic medium to eliminate plasmalema and cytoplasmic content. Lactate respiration was sensitive to respiratory chain effectors, such as oligomycin and antimycin. To evaluate the LDH-X contribution to mitochondrial respiration, lipoate dehydrogenase was inhibited through 5-methoxyindole-2-carboxylic acid (MICA) in the presence of pyruvate-malate and citrate-malate, obtaining with the addition of lactate, oxygen uptakes of 18% and 51% with respect to respiration with the mentioned substrates. In the MICA dose-effect curve, a major sensitivity to inhibitor in active state mitochondrial respiration is obtained when pyruvate-malate is used. Lactate competence with pyruvate by mitochondrial LDH-X was observed. The results obtained would allow the thorough study of the necessity of oxidative energy in the capacitation and fertilization processes, and of the LDH-X role in frozen-stored bovine sperm.     

Control of respiration and oxidative phosphorylation in isolated rat liver cells

NAD(P)H fluorescence, mitochondrial membrane potential and respiration rate were measured and manipulated in isolated liver cells from fed and starved rats in order to characterize control of mitochondrial respiration and phosphorylation. Increased mitochondrial NADH supply stimulated respiration and this accounted for most of the stimulation of respiration by vasopressin and extracellular ATP. From the response of respiration to NADH it was estimated that the control coefficient over respiration of the processes that supply mitochondrial NADH was about 0.15-0.3 in cells from fed rats. Inhibition of the ATP synthase with oligomycin increased the mitochondrial membrane potential and decreased respiration in cells from fed rats, while the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone had the opposite effect. There was a unique relationship between respiration and membrane potential irrespective of the ATP content of the cells indicating that phosphorylation potential controls respiration solely via phosphorylation (rather than by controlling NADH supply). From the response of respiration to the mitochondrial membrane potential (delta psi M) it was estimated that the control coefficients over respiration rate in cells from fed rats were: 0.29 by the processes that generate delta psi M, 0.49 by the process of ATP synthesis, transport and consumption, and 0.22 by the processes that cycle protons across the inner mitochondrial membrane other than via ATP synthesis (e.g. the passive proton leak). Control coefficients over the rate of mitochondrial ATP synthesis were 0.23, 0.84 and -0.07, respectively, by the same processes. The control distribution in cells from starved rats was similar.     

Effects of Ultraviolet Radiation on Respiration and Growth in Radiation-resistant and Radiation-sensitive Strains of Escherichia coli B

Ultraviolet (UV) irradiation at 254 nm causes different respiration and growth responses in log-phase cultures of Escherichia coli B/r and B(s-1). These differences are correlated with the ability and inability, respectively, of these bacterial strains to repair UV-induced lesions in deoxyribonucleic acid (DNA). After irradiation, B(s-1) cells (radiation-sensitive) exhibit uncoupling of growth and respiration; growth and synthesis cease, whereas respiration continues. B/r cells (radiation-resistant) grown on glycerol exhibit severe temporary inhibition of growth and respiration after UV, and the coupling of these two processes is maintained, except at a very high UV dose. Inhibition begins at about the time DNA synthesis resumes and continues for a period of time that is dependent upon dose. Glucose-grown cells do not exhibit severe respiratory, growth, and synthetic inhibitions; these processes remain coupled in the cells during the postirradiation period. Photoreactivation treatment delays uncoupling of growth and respiration in B(s-1) and prevents inhibition of respiration and growth in B/r. These results indicate that the postirradiation responses result from the presence of pyrimidine dimers in DNA. Ultraviolet irradiation of B/r and B(s-1) cells results in an accumulation of adenosine triphosphate by 30 min after UV. This accumulation decreases with time and does not appear to be related to the inhibition of respiration in glycerol-grown B/r cells. The results on B/r are interpreted in terms of a control mechanism for reestablishment of a balance among macromolecules in the irradiated cells so as to provide them with the potential to survive. The specific steps in such a reestablishment of balance appear to depend upon the substrate oxidized. In B(s-1) cells, which cannot repair UV-induced damage in DNA, some control mechanism that coordinates cellular processes may be inactivated.