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PubMed1993

Characterization of morphology and cellular metabolism during the spheroid formation by fibroblasts.

Normal adult human dermal fibroblasts were cultured at 37 degrees C on a thermoresponsive substratum composed of poly-N-isopropyl acrylamide (PNIPAAm) and type I collagen. The confluent fibroblasts were forced to detach from the substratum as a monolayer cell sheet made of polygonal cells linked together with many microvilli by decreasing an ambient temperature from 37 degrees C to a temperature below the lower critical solution temperature of PNIPAAm (about 30 degrees C). The floating cell sheet shrank and condensed into an aggregate with gap and tight junctions within several hours and finally formed a spheroid by 2 days. Spheroids were covered with squamous cells and contained cuboidal cells inside. Cycloheximide and actinomycin D reversibly inhibited the spheroid formation. Extracellular matrix fibrils deposited among inner cells when the culture period extended over 7 days. A 28-day-old spheroid with a diameter of about 600 microns contained live cells even in the central region. Cellular metabolic activities such as glucose consumption, lactic acid production, and ATP content of spheroids were significantly lower than those of monolayers. The spheroid described in the present study seems to be a useful in vitro model of connective tissues.

Adenosine Triphosphate↗
PubMed1975

The extent of the stimulated electrical potential decay under phosphorylating conditions and the H+/ATP ratio in Rhodopseudomonas sphaeroides chromatophores following short flash excitation.

1. In chromatophores from Rps. sphaeroides, the stimulation by ADP and Pi of the electric potential decay indicated by the carotenoid shift is greater than the stimulation of the decay of pH change indicated by the colour change of added cresol red under similar conditions. This difference is attributed to H+ consumption during the synthesis of ATP. The ratio of H+ translocated across the membrane to ATP synthesized was estimated to be approximately 1.7 H+/ATP. 2. The stimulation of the electrical potential decay by ADP and Pi was found to be a constant fraction (10%) of the total decay when the flash intensity was varied. No 'critical' or 'threshold' potential was observed. 3. The stimulated electrical potential decay after a second flash, given within a few seconds of the first, was related to the amplitude of the electrical potential produced by the second flash (10%) but neither to the dark time between the flashes, nor to the total extent of the electrical potential above the dark level. These results are consistent with two hypotheses (a) the chromatophores are a mixed population of vesicles, only a small fraction (10%) of which possess an active ATP synthesizing system (b) the activity of the ATP synthesizing system, though driven by a proton motive force, is controlled by electron transport processess. If alternative (a) is correct then the overall single turnover flash yield of 1 ATP per 1470 bacteriochlorophyll measured in (1) would mean that the yield of the active vesicles is approximately 10 ATP per 1470 bacteriochlorophyll or 30 ATP per vesicle. 4. The stimulation of the electrical potential decay by ADP and Pi is approximately 40% less in antimycin-treated chromatophores. It is shown that this is probably a consequence of antimycin-inhibited H+-release on the inside of the chromatophore vesicles following a flash.

Adenosine Diphosphate↗
PubMed2006

A necrotic cell death model in a protist.

While necrotic cell death is attracting considerable interest, its molecular bases are still poorly understood. Investigations in simple biological models, taken for instance outside the animal kingdom, may benefit from less interference from other cell death mechanisms and from better experimental accessibility, while providing phylogenetic information. Can necrotic cell death occur outside the animal kingdom? In the protist Dictyostelium, developmental stimuli induced in an autophagy mutant a stereotyped sequence of events characteristic of necrotic cell death. This sequence included swift mitochondrial uncoupling with mitochondrial 2',7'-dichlorofluorescein diacetate fluorescence, ATP depletion and increased oxygen consumption. This was followed by perinuclear clustering of dilated mitochondria. Rapid plasma membrane rupture then occurred, which was evidenced by time-lapse videos and quantified by FACS. Of additional interest, developmental stimuli and classical mitochondrial uncouplers triggered a similar sequence of events, and exogenous glucose delayed plasma membrane rupture in a nonglycolytic manner. The occurrence of necrotic cell death in the protist Dictyostelium (1) provides a very favorable model for further study of this type of cell death, and (2) strongly suggests that the mechanism underlying necrotic cell death was present in an ancestor common to the Amoebozoa protists and to animals and has been conserved in evolution.

Animals↗
PubMed1984

cis-Unsaturated fatty acids uncouple mitochondria and stimulate glycolysis in intact lymphocytes.

In intact lymphocytes, linoleic acid acts as a typical mitochondrial uncoupler: it substantially decreases the mitochondrial membrane potential and the cellular ATP level, while stimulating oxygen consumption and glycolysis. Under these conditions the inhibition of cellular functions by linoleic acid cannot be attributed to its postulated effects on lipid domains in the plasma membrane.

Adenosine Triphosphate↗
PubMed2001

The effect of two additive solutions on the postthaw storage of RBCs.

BACKGROUND: Sterile systems for freezing and for washing thawed blood will allow the storage of RBCs for more than 24 hours after removal of the cryoprotectant glycerol. This study assessed the effect of two ASs in maintaining deglycerolized RBCs. STUDY DESIGN AND METHODS: Twenty-four RBC units were stored for 6 days, pooled in groups of 4, realiquoted, sterilely glycerolized, and frozen. One month later, the units were thawed, sterilely deglycerolized by using an automated system (H215; Haemonetics), and stored for 5 weeks in either 100 or 200 mL of AS-3 or an experimental AS (EAS-61). Sterile samples were taken weekly for chemical and morphometric analysis. RESULTS: The glycerolization and deglycerolization process produced highly comparable RBC units, but it caused a marked reduction of RBC pH, to about 6.4 at the beginning of storage. The addition of acidic AS-3 further reduced the pH, which in turn reduced glucose consumption, lactate formation, and RBC ATP concentrations. Alkaline EAS-61 increased these measures. Hypotonic EAS-61 caused increased cell swelling and hemolysis, despite better RBC morphology. CONCLUSIONS: Automation of sterile glycerolization and deglycerolization with the H215 works well, but the solutions should be reformulated for extended postthaw storage. This would best be accomplished by raising the pH of the wash solutions by the addition of disodium phosphate or sodium bicarbonate or both, by using alkaline ASs, and by matching the osmolality of the wash solution and ASs.

Adenine↗
PubMed1989

Respiratory burst oxidase of fertilization.

Partially reduced oxygen species are toxic, yet sea urchin eggs synthesize H2O2 in a "respiratory burst" at fertilization, as an extracellular oxidant to crosslink their protective surface envelopes. To study the biochemical mechanism for H2O2 production, we have isolated an NADPH-specific oxidase fraction from homogenates of unfertilized Strongylocentrotus purpuratus eggs that produces H2O2 when stimulated with Ca2+ and MgATP2-. Concentrations of free Ca2+ previously implicated in regulation of egg activation modulate the activity of the oxidase. Inhibitors were used to test the relevance of this oxidase to the respiratory burst of fertilization. Procaine, two phenothiazines, and N-ethylmaleimide (but not iodoacetamide) inhibited H2O2 production by the oxidase fraction and oxygen consumption by activated eggs. The ATP requirement suggested that protein kinase activity might regulate the respiratory burst of fertilization; consonant with this hypothesis, H-7 and staurosporine were inhibitory. The respiratory burst oxidase of fertilization is an NADPH:O2 oxidoreductase that appears to be regulated by a protein kinase; although it bears a remarkable resemblance to the neutrophil oxidase, unlike the latter it does not form O2- as its initial product.

Animals↗
PubMed1999

Na+ pump inhibition and non-selective cation channel activation by cyanide and anoxia in guinea-pig chromaffin cells.

1. Hypoxia and metabolic inhibition with cyanide (CN) evoke catecholamine secretion in adrenal chromaffin cells through depolarization. We elucidated mechanisms for a CN- or anoxia-induced inward (depolarization) current, using the perforated patch method. 2. Bath application of Ba2+ induced a dose-dependent inhibition of a muscarine-induced current (IMUS) and part of the CN-induced current (ICN) with an IC50 (concentration responsible for 50 % inhibition) of 1.3 mM. The Ba2+-sensitive component was estimated to comprise 58 % of the total ICN. 3. The Ba2+-resistant component of ICN tended to increase with shifts of membrane potential from -40 to 40 mV and was markedly suppressed by exposure to a K+-free solution or 200 microM ouabain, indicating that the majority of the Ba2+-resistant component of ICN is due to suppression of the Na+ pump current (Ipump). 4. The non-Ipump component of ICN diminished progressively in K+-free solution. Substitution of glucose for sucrose in a K+-free CN solution further diminished the CN potency to produce the non-Ipump component. 5. The I-V relationship for the non-Ipump component of ICN had a reversal potential of -3 and -47 mV at 147 and 5.5 mM Na+, respectively, and showed an outward rectification, indicating that the non-Ipump component of ICN is due to activation of non-selective cation channels. 6. Exposure to anoxia induced a current with an amplitude comparable to that of ICN, and the anoxia-induced current apparently occluded development of ICN. The anoxia-induced current diminished by ca 60 % in the absence of K+ and reversed polarity at 5 mV under K+-free conditions. 7. It is concluded that exposure to CN and to anoxia induces suppression of the Na+ pump and activation of non-selective cation channels, probably due to an ATP decrease resulting mainly from consumption by the Na+ pump.

Animals↗
PubMed2005

Activation of hypoxia-inducible factor-1 in bacillary angiomatosis: evidence for a role of hypoxia-inducible factor-1 in bacterial infections.

BACKGROUND: Bartonella species are the only known bacterial pathogens causing vasculoproliferative disorders in humans (bacillary angiomatosis [BA]). Cellular and bacterial pathogenetic mechanisms underlying the induction of BA are largely unknown. METHODS AND RESULTS: Activation of hypoxia-inducible factor-1 (HIF-1), the key transcription factor involved in angiogenesis, was detected in Bartonella henselae-infected host cells in vitro by immunofluorescence, Western blotting, electrophoretic mobility shift, and reporter gene assays and by immunohistochemistry in BA tissue lesions in vivo. Gene microarray analysis revealed that a B henselae infection resulted in the activation of genes typical for the cellular response to hypoxia. HIF-1 was essential for B henselae-induced expression of vascular endothelial growth factor as shown by inhibition with the use of HIF-1-specific short-interfering RNA. Moreover, infection with B henselae resulted in increased oxygen consumption, cellular hypoxia, and decreased ATP levels in host cells. Infection with a pilus-negative variant of B henselae did not lead to cellular hypoxia or activation of HIF-1 or vascular endothelial growth factor secretion, suggesting a crucial role of this bacterial surface protein in the angiogenic reprogramming of the host cells. CONCLUSIONS: B henselae induces a proangiogenic host cell response via HIF-1. Our data provide for the first time evidence that HIF-1 may play a role in bacterial infections.

Adenosine Triphosphate↗
PubMed2003

Enhanced intracellular calcium promotes metabolic and secretory disturbances in rat gastric mucosa during ethanol-induced gastritis.

Changes in the Ca(2+) homeostasis have been implicated in cell injury and death. However, Ca(2+) participation in ethanol-induced chronic gastric mucosal injury has not been elucidated. We have developed a model of ethanol-induced chronic gastric injury in rats, characterized by marked alterations in plasma membranes from gastric mucosa and a compensatory cell proliferation, which follows ethanol withdrawal. Therefore, the present study explored the possible role of intracellular Ca(2+) in the oxidative metabolism and in acid secretion in this experimental model. Glucose oxidation was greatly enhanced in the injured mucosa, as evaluated by CO(2) production by isolated mucosal preparations incubated with (14)C-radiolabeled glucose in different carbons. Oxygen consumption and acid secretion (aminopyrine accumulation) were also stimulated. A predominating secretory status was morphologically identified by electron microscopy in oxyntic cells of gastric mucosa from ethanol-treated rats. A coupling between secretory and metabolic effects induced by ethanol (demonstrated by an inhibitory effect of omeprazole in both parameters) was found. These ethanol-induced effects were also inhibited by addition of Ca(2+) chelators to isolated gastric mucosa samples. Lanthanum, a Ca(2+) channel blocker, inhibited ethanol-promoted increase of oxidative metabolism. In addition, a stimulated Ca(2+) uptake by mucosal minces and increased in vivo Ca(2+) levels in cytosolic and mitochondrial fractions, were also noticed. Enhanced glucose and oxygen consumptions were associated with higher ATP and NADP+ availability, whereas cytosolic NAD/NADH ratio (assessed by mucosal levels of lactate and pyruvate) was not significantly modified by the chronic ethanol administration. In conclusion, changes in Ca(2+) homeostasis, probably mainly due to increased extracellular Ca(2+) uptake, could mediate secretory and metabolic alterations found in the gastric mucosa from rats chronically treated with ethanol.

Adenosine Triphosphate↗
PubMed1998

Dependence of energy metabolism on the density of cells in culture.

Metabolism in cultured cells strongly depends on cell density, thus cell density may be an important factor in biotechnological maintenance of cell cultures. Therefore the energy metabolism of three related cell types with different proliferation activity has been characterised: density controlled primary or secondary fibroblasts [pmf], immortalized, but still density controlled 3T3 cells, and SV40-transformed 3T3 lacking growth control. The investigations revealed the decrease of oxygen consumption, net lactate production, ATP-content, NAD-content and NAD-redox potential, and F-actin content with increasing culture density in pmfs, less distinct in 3T3 cells and in SV40-3T3 cells. The main decrease of these factors is related to cell-cell contacts rather than to proliferation which ceases at least two division cycles after most cells contacted a neighbouring cell. SV40-3T3 cells also at preconfluent densities exhibit relatively low metabolic activity as revealed by the above mentioned factors. Supply with metabolites for catalytic processes seems to be the rate limiting factor as deduced from a decreasing NADH/total NAD ratio. SV40-3T3 cells lack a contact mediated reduction of energy metabolism which is in accord with the missing contact inhibition of motility and proliferation in SV40-3T3 cells. Because of a possible association of glycolytic enzymes with actin, F-actin content has been determined. No correlation, however, was revealed by the F-actin-/lactate ratio. This may be due to the fact that about 50% of the lactate released into the culture medium originated to from glutaminolysis rather than glycolysis. Only in SV40-3T3 cells were respiration and lactate production insensitive to glutamine deprivation and in these cells both these parameters did not change significantly, therefore they did not allow testing as to whether f-actin content and glycolytic activity are correlated. Fractionated release of adeninnucleotides revealed that energy charge in the cytosolic fraction is the only factor which strictly correlates with the cessation of proliferation. Thus energy charge of the cytosol seems to be the only factor of these studied here, which can be used as an indicator of a culture's commitment for further proliferation. To compare energy metabolic parameters among different cell types one always has to take into account the density dependence of these factors.

3T3 Cells↗
PubMedDate not supplied

Enzyme-linked immunosorbent assay for measurement of JNK, ERK, and p38 kinase activities.

A rapid enzyme-linked immunosorbent assay for the enzyme activity measurement of three well-known mitogen-activated protein (MAP) kinases, JNK2, ERK2, and p38 is described. The assay involves immobilization of the respective kinase substrates c-Jun, Elk1, or ATF2 on microtiter plates, addition of the kinase reaction mixture, and measurement of substrate phosphorylation using phospho-epitope-specific antibodies. This novel procedure represents a marked improvement to conventional radioactive MAP kinase assays in terms of quantification, precision, performance at physiological ATP concentration, high throughput, time consumption and amenability to automation. In addition to the standard solid phase assay using plastic-bound protein substrates, we developed an alternative solution phase protocol using soluble protein substrates. By comparing the results of the two assays, we found that MAP kinases retained much of their substrate specificity in the phosphorylation of immobilized protein substrates. Interestingly, we observed a strong preference of JNK2 and p38 for the phosphorylation of dimeric over monomeric substrates. We further characterized the kinase inhibitory activity of olomoucine, staurosporine, and SB 203580 for JNK2, ERK2, and p38. Taken together, this assay could assist in the biochemical characterization of MAP kinases and in identifying potent and specific inhibitors of these enzymes.

3T3 Cells↗
PubMedDate not supplied

[The effect of verapamil on the dynamics of decrease in the brain levels of phosphorus macroergs during ischemia studied by 31P-NMR in vivo].

Protective effects of verapamil on dynamics of phosphorus-containing metabolites were studied during 30 min complete ischemia by means of 31P NMR. Verapamil appears to decrease the ATP and creatine phosphate pools consumption in ischemic brain tissue. The efficiency of the drug depended on the administration procedure and was not similar in two different models of ischemia. Possible mechanisms of the verapamil effect on bioenergetics of nervous tissue are discussed.

Adenosine Triphosphate↗
PubMedDate not supplied

[Characteristics of the stoichiometric regulation of glycolysis in prokaryotic cells. A model].

A model is developed to investigate the steady state of the glycolytic system of procaryotic cells which have the hexose transport into the cell coupled with phosphoenolpyruvic acid-dependent phosphorylation. Analysis of the model shows that this phosphorylation results: 1) in stoichiometric connection between the rate of hexose consumption and the utilization of ATP and 2) the stoichiometric positive feedback. The latter can be the cause of bistability, oscillations and other nonlinear phenomena.

Bacteria↗
PubMed1984

Efficacies of whole blood and plasma cross circulation on galactosamine induced hepatic failure rats.

Galactosamine induced hepatic failure rats were treated by whole blood and plasma cross circulation. While no significant improvement in survival rate was achieved by means of these 2 procedures, oxygen consumption in state 3 and ATP synthesis of mitochondrial function were significantly improved as compared to the sham circulation groups. Maintenance of mitochondrial functions as by extracorporeal perfusion techniques may be advantageous in acute hepatic failure.

Animals↗
PubMed1991

Dependence of cerebral energy phosphate and evoked potential recovery on end-ischemic pH.

We determined whether the rate of metabolic recovery and electrophysiological deficit after incomplete cerebral ischemia is related to intracellular pH (pHi) achieved at the end of ischemia in a dose-dependent manner. End-ischemic pHi was varied by employing two ischemic durations, 12 and 30 min, and by setting preischemic plasma glucose to approximately 80 or 400 mg/dl. Incomplete global ischemia was produced in anesthetized dogs by transient intracranial hypertension followed by 4 h of reperfusion, and pHi, ATP, and phosphocreatine (PCr) were measured with 31P magnetic resonance spectroscopy. Cerebral blood flow was reduced to approximately 6 ml.min-1.100 g-1 during ischemia. End-ischemic pHi was greater than 5.7 in all animals from various treatment groups except for four of seven dogs treated with 30-min hyperglycemic ischemia. When end-ischemic pHi remained greater than 5.7, there was nearly complete recovery of ATP, PCr, pHi, intracellular bicarbonate concentration [( HCO3-]i), and O2 consumption. Partial recovery of somatosensory-evoked potentials (SEP) occurred in most of these animals. In the 30-min hyperglycemic animals in which pHi fell below 5.5, ATP, PCr, and O2 consumption recovered by only one-half over 60 min of reperfusion and then declined to near-zero levels without SEP recovery. In addition, pHi remained less than 6.0, and [HCO3-]i remained less than 2 mM throughout reperfusion. We conclude that there is an apparent in vivo pHi threshold of approximately 5.5-5.7 during incomplete cerebral ischemia that is associated with an inability to significantly restore pHi and [HCO3-]i and with secondary deterioration of high-energy phosphate levels.

Animals↗
PubMed1993

The quantitative contributions of mitochondrial proton leak and ATP turnover reactions to the changed respiration rates of hepatocytes from rats of different thyroid status.

Hepatocytes from hypothyroid rats respire more slowly than those from control animals; cells from hyperthyroid rats respire faster. We have identified the blocks of reactions whose kinetics are significantly affected by thyroid hormones and have quantified the contribution of flux through different blocks of energy-dissipating reactions to the altered oxygen consumption. In cells from hypothyroid rats compared with littermate euthyroid controls, there were significant kinetic effects on non-mitochondrial oxygen consumption reactions and on mitochondrial proton leak but not on ATP turnover. Approximately 50% of the decrease in cellular oxygen consumption of hypothyroid cells was accounted for by a decrease in mitochondrial proton leak rate and approximately 50% by decreased non-mitochondrial oxygen consumption. Metabolic control analysis showed that the distribution of control over oxidative phosphorylation and mitochondrial potential in hepatocytes from hypothyroid animals was broadly similar to its distribution in euthyroid controls. In cells from hyperthyroid rats compared with littermate euthyroid controls, there were significant kinetic effects on mitochondrial proton leak and on the reactions involved in ATP synthesis and consumption but not on non-mitochondrial oxygen consumption. Approximately 50% of the increase in cellular oxygen consumption in hyperthyroid cells was accounted for by an increased mitochondrial proton leak rate and the remainder by increased ATP turnover; there were no changes in non-mitochondrial oxygen consumption.

Adenosine Triphosphate↗
PubMed1987

Yeast diadenosine 5',5'''-P1,P4-tetraphosphate alpha,beta-phosphorylase behaves as a dinucleoside tetraphosphate synthetase.

The diadenosine 5',5'''-P1,P4-tetraphosphate alpha,beta-phosphorylase (Ap4A phosphorylase), recently observed in yeast [Guaranowski, A., & Blanquet, S. (1985) J. Biol. Chem. 260, 3542-3547], is shown to be capable of catalyzing the synthesis of Ap4A from ATP + ADP, i.e., the reverse reaction of the phosphorolysis of Ap4A. The synthesis of Ap4A markedly depends on the presence of a divalent cation (Ca2+, Mn2+, or Mg2+). In vitro, the equilibrium constant K = ([Ap4A][Pi])/[(ATP][ADP]) is very sensitive to pH. Ap4A synthesis is favored at low pH, in agreement with the consumption of one to two protons when ATP + ADP are converted into Ap4A and phosphate. Optimal activity is found at pH 5.9. At pH 7.0 and in the presence of Ca2+, the Vm for Ap4A synthesis is 7.4 s-1 (37 degrees C). Ap4A phosphorylase is, therefore, a valuable candidate for the production of Ap4A in vivo. Ap4A phosphorylase is also capable of producing various Np4N' molecules from NTP and N'DP. The NTP site is specific for purine ribonucleotides (N = A, G), whereas the N'DP site has a broader specificity (N' = A, C, G, U, dA). This finding suggests that the Gp4N' nucleotides, as well as the Ap4N' ones, could occur in yeast cells.

Acid Anhydride Hydrolases↗
PubMed1993

Catecholamines: important factors in the increase of oxidative phosphorylation coupling in rat-liver mitochondria during the early phase of burn injury.

Thirty minutes after Sprague-Dawley rats had been injected subcutaneously with epinephrine or norepinephrine, the respiratory control ratio (RCR), the rate of O2 consumption in state 3 and the rate of ATP formation in liver mitochondria succinate respiratory chain were increased. The rate of O2 consumption in state 4 was increased or only slightly increased. When the rats were injected with the adrenergic blocking agents, timolol or phenoxybenzamine, 1 h before the burn, the activities of the succinate respiratory chain was partly inhibited 30 min postburn. Compared with our previous results, it can be suggested that catecholamines may play an important role in the increase of oxidative phosphorylation coupling in the early phase of burn injury.

Animals↗