Periodic fluctuations in oxygen consumption comparing HeLa (cancer) and CHO (non-cancer) cells and response to external NAD(P)+/NAD(P)H

Oxygen consumption in the presence of cyanide was utilized as a measure of plasma membrane electron transport in Chinese hamster ovary (CHO) and human cervical carcinoma (HeLa) cell lines. Both intact cells and isolated plasma membranes carry cyanide-insensitive NADH(P)H oxidases at their external membrane surfaces (designated ECTO-NOX proteins). Regular oscillatory patterns of oxygen consumption with period lengths characteristic of those observed for rates of NADH oxidation by ECTO-NOX proteins were observed to provide evidence for transfer of protons and electrons to reduce oxygen to water. The oscillations plus the resistance to inhibition by cyanide identify the bulk of the oxygen consumption as due to ECTO-NOX proteins. With intact CHO cells, oxygen consumption was enhanced by but not dependent upon external NAD(P)H addition. With intact HeLa cells, oxygen consumption was inhibited by both NADH and NAD⁺ as was growth. The results suggest that plasma membrane electron transport from internal donors to oxygen as an external acceptor is mediated through ECTO-NOX proteins and that electron transport to molecular oxygen may be differentially affected by external pyridine nucleotides depending on cell type.     

Oxygen diffusion in large single-celled organisms

The functional relationship between the oxygen uptake rate of a spherical, single cell organism and the external oxygen tension is shown to be related to the dependence of the specific oxygen consumption rate, that is, the consumption rate of an infinitesimal volume element of cellular material, on the external oxygen tension. Analytical solutions of the governing steady state diffusion equation are obtained by dividing the system into three regions, an inner region of the sphere in which oxygen consumption rate depends upon oxygen tension, an outer region of the sphere in which oxygen consumption rate is constant (independent of oxygen tension), a nonconsuming membrane over the sphere that offers only resistance to oxygen diffusion, and an infinite region outside the sphere and membrane supplying oxygen to the system. The solutions show the oxygen tension as a function of position inside the spherical cell for a variety of system parameters.     

A multifunctional hydroquinone oxidase of the external cell surface and sera.

A multifunctional cell surface protein with NADH oxidase (NOX) activity and capable of oxidizing hydroquinones is located at the exterior of the cell and is shed in soluble form into sera. The oxidase appears to function as a terminal oxidase of a trans plasma membrane electron transport chain consisting of a NAD(P)H-ubiquinone reductase at the cytosolic membrane surface, possibly a b-type cytochrome, ubiquinone and the oxidase. Hyperactivity or conditions that interrupt ordered 2H+ + 2e- transport from NAD(P)H or hydroquinone to molecular oxygen and other acceptors at the external cell surface may result in the generation of superoxide. The latter may serve to propagate aging-related redox changes both to adjacent cells and circulating blood components. A circulating NOX activity form associated with aging and the reduction of cytochrome c by sera of aged patients that is partially inhibited by ubiquinone are described.     

The autoregulation of intracellular oxygen tension and the modification of cellular radiosensitivity. The development of an adaptation hypothesis

A hypothesis is reported postulating that the range within which the intracellular oxygen content varies with changes in the external oxygenation conditions is responsible for the oxygen-dependent radiosensitivity modification that may be controlled by the diffusion resistance of a cytoplasmic membrane. The adaptation mechanism of intracellular Po2 autoregulation is involved when drastic changes in the oxygen content of the environment occur. As the oxygen content decreases this mechanism provides maximal values of the intracellular Po2 required for optimizing cell viability; the increased oxygen content prevents cells from oxygen intoxication.     

Endocellular regulation of oxygen consumption in the neurone of isolated crawfish stretch receptor

Morphological correlations of functional regulation of oxygen consumption have been investigated in single of isolated crustacean stretch receptor neuron. The increase in oxygen consumption is promoted by: 1) redistribution of mitochondria and increase in cytochrome oxidase (CO) activity in mitochondria near to the plasmatic membrane; 2) coordination of mitochondria aggregation rhythms with pO2 rhythms in external environment of a cell; 3) reduction of the area with high CO and mitochondria activity, and reduction of the way of oxygen diffusion; 4) increase in CO activity gradient from periphery to the center of the neuron body; 5) carry of oxygen by water current under hydration of the neuron body, and cytoplasm dilution under transition of a part of gel in sol; 6) cyclic changes in the neuron body and hillock sizes ratio determining carry of oxygen by water current into the neuron body, oxygen absorption by mitochondria in the neuron body, and transition of the water released from oxygen from the neuron body into hillock and further into the external environment.     

Intracellular regulation of oxygen consumption in the isolated crayfish stretch receptor neuron

Morphological correlations of the functional regulation of oxygen consumption have been studied on single isolated crayfish mechanoreceptor neurons. An enhancement of oxygen consumption is promoted by the following: (1) redistribution of mitochondria and an increase in cytochrome oxidase (CO) activity in mitochondria near the plasmatic membrane, (2) coordination of mitochondria aggregation rhythms with pO₂ rhythms in the medium external for a cell, (3) a decrease in the area of high CO activity and mitochondria and a shortening of the oxygen diffusion pathway, (4) an increase of the CO activity gradient from the neuron body periphery to its center, (5) a transfer of oxygen with the water flow during neuron body hydration and cytoplasm dilution during the transfer of a portion of the gel into sol, (6) cyclic changes in the ratio of the neuron body and hillock sizes at which there is a transfer of oxygen with the water flow into the neuron body, its mitochondrial uptake in the neuron body, and transfer of the oxygen-free water from the neuron body into the axonal hillock and further into the external medium.     

Relation between the reaction cytochrome oxidase-oxygen and oxygen uptake in cells in vivo. The role of diffusion

Before reaching the oxidase located inside the cell on the mitochondrial membrane, oxygen may be slowed down by diffusion within the cytoplasm. Diffusion or enzymic activity may predominate and this is related to the size and morphology of the organism, the intracellular diffusion coefficient, and the K_m of the oxidizing terminal enzyme. This K_m may be apparently diminished by inhibitors. Different experimental situations can arise in some plants (where oxygen diffusion in bulk tissue is not important in comparison to that in cell): either oxygen diffusion is limiting, or diffusion and an enzymic reaction compete, or diffusion does not slow down the respiratory rate. A mathematical model relates the oxygen concentration in the external medium, to the rate of oxygen uptake at the oxygen cytochrome-oxidase reaction level.     

Respiration-Linked Proton Transport, Changes in External pH, and Membrane Energization in Cells of Escherichia coli

The kinetics of respiration-dependent proton efflux and membrane energization have been studied in intact cells of logarithmic-phase Escherichia coli. Parallel measurements of the rate and extent of proton efflux into the external medium (half-time, about 10 s; ratio of H(+) to O, about 0.5) and the oxidation of E. coli cytochrome b (half-time, 相似文献   

An electrophysiological study of the non-junctional membrane of epidermal cells in Tenebrio molitor

The contribution of K and Cl to the membrane potential of the epidermal cells of the recently-ecdysed larva of the mealworm was examined. The ionic basis for the membrane potential is complex. Although increasing the external K level depolarized the cell membrane, the relationship obtained suggests that ions other than K contribute largely to the recorded membrane potential. In particular, exposing the cells to K concentrations below the normal level of 40 mM has only slight effects on membrane potential, irrespective of whether K is lowered by direct substitution with Na or under conditions in which Na and Cl levels are held constant. Increasing the external Cl levels from 4 mM to 154 mM while holding K and Na levels constant resulted in a 10 mV hyperpolarization. The slight hyperpolarizing effects of high external Cl could be mimicked by citrate, but not by acetate, the latter drastically hyperpolarizing the cell membrane at levels of K that normally maintain a reduced membrane potential. External Na has little effect on the membrane potential at normal physiological levels of K, but may depolarize the cell at low K levels. The results suggest that several inorganic ions, and possibly organic acids, participate in generating the membrane potential of the epidermal cell. The passive ionic properties of non-junctional epidermal membrane and muscle membrane appear to the similar in this insect.The electrical resistance on the non-junctional membrane is highly dependent on the external K level, and can be reduced by three orders of magnitude by increasing external K from 1 mM to 120 mM. The resistance of the junctional membrane remains constant over this range of external K concentrations.     

Porin and cytochrome oxidase containing contact sites involved in the oxidation of cytosolic NADH

Cytochrome c (cyto-c) added to isolated mitochondria promotes the oxidation of extra-mitochondrial NADH and the reduction of molecular oxygen associated to the generation of an electrochemical membrane potential available for ATP synthesis. The electron transport pathway activated by exogenous cyto-c molecules is completely distinct from the one catalyzed by the respiratory chain. Dextran sulfate (500 kDa), known to interact with porin (the voltage-dependent anion channel), other than to inhibit the release of ATP synthesized inside the mitochondria, greatly decreases the activity of exogenous NADH/cyto-c system of intact mitochondria but has no effect on the reconstituted system made of mitoplasts and external membrane preparations. The results obtained are consistent with the existence of specific contact sites containing cytochrome oxidase and porin, as components of the inner and the outer membrane respectively, involved in the oxidation of cytosolic NADH. The proposal is put forward that the bi-trans-membrane electron transport chain activated by cytosolic cyto-c becomes, in physio-pathological conditions: (i) functional in removing the excess of cytosolic NADH; (ii) essential for cell survival in the presence of an impairment of the first three respiratory complexes; and (iii) an additional source of energy at the beginning of apoptosis.     

Substrate binding site for nitrate reductase of Escherichia coli is on the inner aspect of the membrane.

Escherichia coli grown anaerobically on nitrate exhibited the same transport barrier to reduction of chlorate, relative to nitrate, as that exhibited by Paracoccus denitrificans. This establishes that the nitrate binding site of nitrate reductase (EC 1.7.99.4) in E. coli must also lie on the cell side of the nitrate transporter which is associated with the plasma membrane. Because nitrate reductase is membrane bound, the nitrate binding site is thus located on the inner aspect of the membrane. Nitrate pulse studies on E. coli in the absence of valinomycin showed a small transient alkalinization (leads to H+/NO3- congruent to --0.07) which did not occur with oxygen pulses. By analogy with P. denitrificans, the alkaline transient is interpreted to arise from proton-linked nitrate uptake which is closely followed by nitrite efflux. The result is consistent with internal reduction of nitrate, whereas external reduction would be expected to give leads to H+/NO3-ratios approaching --2.     

Stable hybridoma cultivation in a pilot-scale acoustic perfusion system: long-term process performance and effect of recirculation rate

Perfusion systems have the possibility to be operated continuously for several months. It is important that the performance of the cell retention device does not limit the operation time of a perfusion process used in the production of active pharmaceutical ingredients. Therefore, the aim of this study was to investigate the reliability and long-term stability of an acoustic perfusion process using the 200 L/d BioSep. As the BioSep is an external device, it is possible that dependent on the recirculation rate nutrient gradients occur in the external loop, which could affect the cell metabolism. Therefore, the effect of possible nutrient gradients on cell metabolism, viability and productivity was studied by varying the recirculation rate. In this study, it is shown that a perfusion process using a pilot-scale acoustic cell-retention device (200 L/d) is reliable and simple to operate, resulting in a stable 75-day cultivation of a hybridoma cell line producing a monoclonal antibody. The recirculation rate had a significant effect on the oxygen concentration in the external loop, with oxygen being depleted within the cell-retention device at recirculation rates below 6 m3/m(reactor)3.d (=600 L/d). The oxygen depletion at low circulation rates correlated with a slightly increased lactate production rate. For all other parameters no effect of the recirculation rate was observed, including cell death measured through the release of lactate dehydrogenase and specific productivity. A maximum specific productivity of 12 pg/cell.d was reached.     

Ceruloplasmin stimulates thymidine incorporation by CCL-39 cells in the absence of serum or growth factors

The incorporation of tritiated thymidine into CCL-39 cells grown in the absence of fetal calf serum or other growth factors is greatly increased by low concentrations of ceruloplasmin. The stimulation is greater than observed with serum or thrombin. Addition of serum decreases the thymidine incorporation with ceruloplasmin to the level with serum alone. As with serum, the response to ceruloplasmin is high at both 20% and 1% oxygen, which is consistent with the action of ceruloplasmin as an oxidant with a high affinity for oxygen. Since transplasma membrane electron transport increases cell growth and thymidine incorporation, ceruloplasmin may act as a terminal oxidase for ferrous iron or ascorbate to stimulate transplasma membrane electron transport. The four electron transfer from ceruloplasmin to oxygen to form water will prevent peroxide formation at the cell surface. Alternatively, superoxide formation inside the cell or membrane could employ the superoxide dismutase function of ceruloplasmin to produce peroxide. Either mechanism would be consistent with the previously described stimulation of growth by external oxidants.     

Differential effect of the shape of calcium alginate matrices on the physiology of immobilized neuroblastoma N2a and Vero cells: a comparative study

In order to investigate the effect of cell immobilization in calcium alginate gels on cell physiology, we immobilized Vero or N2a neuroblastoma cells in gels shaped either as spherical beads or as thin membrane layers. Throughout a culture period of 4 weeks cell viability, RNA and cytoplasmic calcium concentration and glutathione accumulation were assayed by fluorescence microscopy after provision of an appropriate dye. Non-elaborate culture conditions were applied throughout the experimental period in order to evaluate cell viability under less than optimal storage conditions. Vero cell proliferation was observed only in spherical beads, while N2a cell proliferation was observed in both configurations until the third week of culture. Increased [Ca2+]cyt could be associated with cell proliferation only when cells were immobilized in spherical beads, while a considerable decrease in the biosynthesis of reduced glutathione and RNA was observed in cells immobilized in thin membrane layers. The observed effects of the shape of the immobilization matrix may be due to differences in external mass transfer resistance. Therefore, depending on cell type, cell proliferation could have been promoted by either increased (Vero) or decreased (N2a) nutrient and oxygen flow to immobilized cells. The results of the present study could contribute to an improvement of immobilized cell sensor storability.     

Studies of macrophage cellular response to the extracellular hydrogen peroxide by tilapia model

Reactive oxygen species (ROS) may act as signaling molecules in the physiology responses and the present study aims to investigate the effect of extracellular hydrogen peroxide on macrophages cellular response. The results obtained in the present study showed that the extracellular hydrogen peroxide affectively alter the membrane potential of the cell membrane and ion exchange channels in the cell membrane through intracellular NAD turnover that may lead to an intracellular calcium ion concentration alteration and subsequently induce the downstream signal activation.     

Scale-up of dialysis fermentation for high cell density cultivation of Escherichia coli.

In dialysis fermentations inhibiting metabolites can be removed from cell suspensions resulting in a prolonged exponential growth phase and higher production yields. Because of successful high cell density cultivations of Escherichia coli in a laboratory dialysis reactor, a scale-up of the process was investigated. To provide sufficient membrane area for dialysis in a technical scale fermenter, an external membrane module was used, that was also applied for oxygen supply to the culture in the external loop. Cultivations with recombinant E. coli K12, with and without induction, in 2- and 300-l reactors were carried out using external modules. Cell densities exceeding 190 g l(-1), previously obtained in laboratory dialysis fermentation, were also produced with external dialysis modules. Protein concentration in a 300-l reactor was increased to the 3.8-fold of industrial fed-batch-fermentations.     

Plasma membrane cholesterol: a possible barrier to intracellular oxygen in normal and mutant CHO cells defective in cholesterol metabolism

The effect of the cholesterol content of the plasma membrane on the intracellular concentration of oxygen in Chinese hamster ovary (CHO) cells and their mutants was investigated by EPR oximetry. Total and free cholesterol content was significantly higher in 25 RA CHO cells as compared to wild-type and M 19 CHO cells, with most of the free cholesterol in normal and mutant CHO cells located in the plasma membrane. The plasma membrane cholesterol content also was altered by various biochemical means, and the effect on the oxygen gradient was studied. Comparing the three cell lines, the gradient was larger with increased content of cholesterol in the plasma cell membrane. This result also is supported by an additional increase in the oxygen gradients with the incorporation of additional cholesterol in the plasma membrane and a decrease in the oxygen gradient when the cholesterol was depleted from the plasma membrane. The results indicate that the concentration of cholesterol in the plasma membrane can be an important factor for the magnitude of the oxygen gradient observed across the cell membrane.     

Trehalase activation in yeasts is mediated by an internal acidification

It has been reported that the addition of glucose, uncouplers and nystatin to yeast cells grown in a sugarfree medium causes trehalase activation; it has been postulated that this activation might be mediated by the depolarization of the plasma membrane. In this article the values of membrane potential and pH gradient across the plasma membrane of Saccharomyces cerevisiae have been determined under the same conditions as those in which trehalase is activated. Membrane potential was evaluated from the distribution of triphenylmethylphosphonium, the pH gradient from the distribution of benzoic acid across the plasma membrane. When the effect of several agents on the two components of the electrochemical proton gradient across the plasma membrane of ethanol-grown yeast cells were studied, under trehalase activation conditions, the following observations were made. (a) The addition of glucose activated trehalase and caused internal acidification of the cells, but had practically no effect on the membrane potential. (b) The addition of 200 mM KCl depolarized the cell membrane but did not affect the internal pH, nor trehalase activity. (c) Although carbonyl cyanide m-chlorophenylhydrazone depolarized the cells at external pH 6.0 and 7.0, it only activated trehalase at an external pH 6.0, leading to the acidification of the internal medium at this pH. (d) Nystatin caused an increase in the triphenylmethylphosphonium accumulation at external pH 6.0 and 7.0, but only activated trehalase at external pH 6.0, causing acidification of the cell interior at this pH. (e) Activation of trehalase was also observed when the internal acidification was caused by addition of a weak acid such as acetate. It is concluded that trehalase activation is mediated by an intracellular acidification and is independent of the membrane potential.     

Early Events Induced by the Elicitor Cryptogein in Tobacco Cells: Involvement of a Plasma Membrane NADPH Oxidase and Activation of Glycolysis and the Pentose Phosphate Pathway

Application of the elicitor cryptogein to tobacco (cv Xanthi) is known to evoke external medium alkalinization, active oxygen species production, and phytoalexin synthesis. These are all dependent on an influx of calcium. We show here that cryptogein also induces calcium-dependent plasma membrane depolarization, chloride efflux, cytoplasm acidification, and NADPH oxidation without changes in NAD+ and ATP levels, indicating that the elicitor-activated redox system, responsible for active oxygen species production, uses NADPH in vivo. NADPH oxidation activates the functioning of the pentose phosphate pathway, leading to a decrease in glucose 6-phosphate and to the accumulation of glyceraldehyde 3-phosphate, 3- and 2-phosphoglyceric acid, and phosphoenolpyruvate. By inhibiting the pentose phosphate pathway, we demonstrate that the activation of the plasma membrane NADPH oxidase is responsible for active oxygen species production, external alkalinization, and acidification of the cytoplasm. A model is proposed for the organization of the cryptogein responses measured to date.     

An alternative hypothesis for the direction of hydrogen ion movement and energy transduction in H. halobium.

It is generally accepted that purple membrane of $\text{H.}\text{halobium}$ functions as a light-driven hydrogen ion pump translocating hydrogen ions from inside the cell to the external medium. However, experimental data from this laboratory together with those obtained by others have always shown an initial alkalinization of the external medium in the light. Additionally, we have found that oxygen can also induce an alkalinization of the bathing solution in the dark. These results can be readily explained if the direction of hydrogen-ion translocation is reversed, that is that both light and oxygen generate an electrochemical gradient of hydrogen ions, which is outwardly directed for ATP synthesis.