Triosephosphates as Intermediates of the Crabtree Effect.

An increase in glucose concentration in the medium rapidly decreases respiration rate in many cell types, including tumor cells. The molecular mechanism of this phenomenon, the Crabtree effect, is still unclear. It was shown earlier that adding the intermediate product of glycolysis fructose-1,6-bisphosphate to isolated mitochondria suppresses their respiration. To study possible roles of glycolytic intermediates in the Crabtree effect, we used a model organism, the yeast Saccharomyces cerevisiae. To have the option to rapidly increase intracellular concentrations of certain glycolytic intermediates, we used mutant cells with glycolysis blocked at different stages. We studied fast effects of glucose addition on the respiration rate in such cells. We found that addition of glucose affected cells with deleted phosphoglycerate mutase (strain gpm1-delta) more strongly than ones with inactivated aldolase or phosphofructokinase. In the case of preincubation of gpm1-delta cells with 2-deoxyglucose, which blocks glycolysis at the stage of 2-deoxyglucosephosphate formation, the effect of glucose addition was absent. This suggests that triosephosphates are intermediates of the Crabtree effect. Apart from this, the incubation of gpm1-delta cells in galactose-containing medium appeared to cause a large increase in their size. It was previously shown that galactose addition did not have any short-term effect on respiration rate of gpm1-delta cells and, at the same time, strongly suppressed their growth rate. Apparently, the influence of increasing triosephosphate concentration on yeast physiology is not limited to the activation of the Crabtree effect.

Negative Feedback of Glycolysis and Oxidative Phosphorylation: Mechanisms of and Reasons for It.

There are two main pathways of ATP biosynthesis: glycolysis and oxidative phosphorylation. As a rule, the two pathways are not fully active in a single cell. In this review, we discuss mechanisms of glycolytic inhibition of respiration (Warburg and Crabtree effects). What are the reasons for the existence of this negative feedback? It is known that maximal activation of both processes can cause generation of reactive oxygen species. Oxidative phosphorylation is more efficient from the energy point of view, while glycolysis is safer and favors biomass synthesis. This might be the reason why quiescent cells are mainly using oxidative phosphorylation, while the quickly proliferating ones - glycolysis.

[Investigation of Mycobacterium tuberculosis sensitivity to fluoroquinolone, by revealing gyrA gene mutations].

The resistance of Mycobacterium tuberculosis (MBT) to fluoroquinolones is associated with the mutations concentrated in the gyrA gene that is a structural gene of a gyrase A subunit. Detection of mutations in this portion of the gene allows the sensitivity of MBT to this group of drugs to be rapidly determined.

Typing of Mycobacterium tuberculosis strains resistant to rifampicin and isoniazid by molecular biological methods.

Mutations in the rpoB, katG, inhA, oxyR/ahpC genes in rifampicin- and isoniazid-resistant M. tuberculosis strains isolated from residents of Moscow, Astrakhan', and Moldova Republic were studied by molecular biological methods (heteroduplex analysis, single strand conformational polymorphism, biochips). Twenty-five combinations of mutations were detected. Some differences in the type distribution of detected mutations were found. The use of biochips is the most perspective method for determining the type of mutation.

Effects of cold exposure in vivo and uncouplers and recouplers in vitro on potato tuber mitochondria.

Effects of cold exposure in vivo and treatment with laurate, carboxyatractylate, atractylate, nucleotides, and BSA in vitro on potato tuber mitochondria have been studied. Cold exposure of tubers for 48-96 h resulted in some uncoupling that could be reversed completely by BSA and partially by ADP, ATP, UDP, carboxyatractylate, and atractylate. UDP was less effective than ADP and ATP, and atractylate was less effective than carboxyatractylate. The recoupling effects of nucleotides were absent when the nucleotides were added after carboxyatractylate. GDP, UDP, and CDP did not recouple mitochondria from either the control or the cold-exposed tubers. This indicates that the cold-induced fatty acid-mediated uncoupling in potato tuber mitochondria is partially due to the operation of the ATP/ADP antiporter. As to the plant uncoupling protein, its contribution to the uncoupling in tuber is negligible or, under the conditions used, somehow desensitized to nucleotides.

[Humoral autoimmune responses to neurospecific proteins in rats with experimental allergic encephalomyelitis and immunomodulation with allogeneic nervous tissue]. / Doslidzhennia humoral'nykh autoimunnykh reaktsii do neirospetsyfichnykh bilkiv u razi eksperymental'noho alerhichnoho entsefalomiielitu na etapakh patohenzu ta imyhokorektsiï alohennoiu nervovoiu tkanynoiu.

Possible immunocorrective effect of the embryonic nervous tissue (NT) and using the model of an experimental allergic encephalomyelitis (EAE) in treating patients with demyelinizing diseases is a prospective field of research. EAE was induced by immunization of rats with the mixture of the spinal cord and BCV at paw pillows. Immunocorrective effect of the allogenic NT was studied in the animals with EAE following the intraperitoneal injection of a newborn rat's NT, namely: enriched fractions of glial and neuronal cell populations. Both intact and EAE animals, treated with culture medium, were used as controls. The clinical signs appeared on 12-th day after EAE inducing. The severe clinical course of EAE was accompanied with statistically significant higher titers of the autoantibodies to MBP as compared with the mild clinical course. The results obtained evidence for the possible immunocorrective effect of enriched fractions of the newborn rat's nervous cells on EAE, and for immunosuppressive effect of the neuronal fraction of NT on the immunopathological processes in EAE.

Effect of the cationic detergent CTAB on the involvement of ADP/ATP antiporter and aspartate/glutamate antiporter in fatty acid-induced uncoupling of liver mitochondria.

The influence of the positively charged amphiphilic compound cetyltrimethyl ammonium bromide (CTAB) on palmitate- and laurate-induced uncoupling and on carboxyatractylate and glutamate recoupling effects in liver mitochondria have been studied. CTAB (40 microM) in the presence of 3 mM MgCl2 had little (if any) effect on the palmitic acid-stimulated respiration of mitochondria; the glutamate recoupling effect increased, and the carboxyatractylate recoupling effect decreased to the same degree with the combined effect (about 80%) remaining unchanged. Thus, CTAB decreases the ADP/ATP antiporter involvement and increases to the same extent the aspartate/glutamate antiporter involvement in the fatty acid-induced uncoupling. The carboxyatractylate and glutamate recoupling effects were less pH dependent in the presence of CTAB than in its absence. These data could be interpreted with the assumption that fatty acid anions are more accessible to the ADP/ATP antiporter and their neutral forms are more accessible to the aspartate/glutamate antiporter, and that CTAB changes the relative anion carrier involvement in the fatty acid-induced uncoupling as it forms neutral complexes with fatty acid anions.

[Modeling of experimental allergic encephalomyelitis in rats and immunomodulation with allogeneic brain cells]. / Modeliuvannia eksperymental'noho alerhichnoho entsefalomiielitu u shchuriv i ioho korektsiia klitynamy alohennoho holovnoho mozku.

We studied the immunocorrective effect of the allogenic new-born brain cells on the model of rats experimental allergic encephalomyelitis (EAE). EAE was induced by the immunization of old rats spinal cord homogenate in Freund's adjuvant. Correction was carried on the 12-th, 14-th, 16-th day after the induction of the EAE by the intraperitoneum injection of rat's new-born brain fractions enriched with neurons and glial cells. A positive clinical effect was achieved by the employment of neurons (the stabilization of encephalomyelitis and the acceleration of the recovery). The glia correction was accompanied by the aggravation in the course of encephalomielitis and by extension of its clinical manifestation period. The obtained results testify to the existence of both an immunoregulative and a neurotrophic influence of the neuron fraction of the new-born brain cells. The mechanism of a corrective effect needs further special investigation.

Comparative study on uncoupling effects of laurate and lauryl sulfate on rat liver and skeletal muscle mitochondria.

Uncoupling effects of laurate and lauryl sulfate have been studied in the isolated rat liver and skeletal muscle mitochondria. In the oligomycin-treated liver mitochondria, 0.02 mM laurate or 0.16 mM lauryl sulfate caused a two-fold stimulation of respiration, accompanied by a membrane potential decrease. Carboxyatractylate (CAtr) and glutamate (or aspartate) strongly decrease the effect of laurate and lauryl sulfate on respiratory rate and membrane potential (the recoupling effect). With both uncouplers, this effect is maximal for CAtr and glutamate (aspartate) at pH 7.8 and 7.0, respectively. Tetraphenyl phosphonium cations, which decrease negative membrane charges, cause an alkaline shift of these pH dependences. Small amounts of lauryl sulfate, which increase the membrane negative charge, induce the opposite shift when laurate is used as an uncoupler. ADP, but not GDP, partially recouple with both laurate and lauryl sulfate. We conclude that lauryl sulfate-induced uncoupling in rat liver, like the uncoupling induced by laurate, is mediated by the ATP/ ADP and glutamate/aspartate antiporters. In skeletal muscle mitochondria uncoupled by laurate, 200 microM GDP causes partial recoupling which can be enhanced by a subsequent additions of CAtr, glutamate and serum albumin. CAtr added before GDP promotes a larger recoupling than when added after GDP and prevents the subsequent effect of GDP. ADP is effective as recoupler at lower concentrations that GDP, whereas CDP is without influence. Lauryl sulfate uncoupling of skeletal muscle mitochondria is GDP-resistant but is sensitive to ADP, CAtr, glutamate and serum albumin. Our data suggest that in skeletal muscle mitochondria a GDP-sensitive mechanism is involved in uncoupling induced by laurate. This mechanism is absent in liver mitochondria. Possible mechanisms of laurate and lauryl sulfate-induced uncoupling are discussed.

[The study of parathyroid hormone in the serum of patient with hypopharyngeal cancer]. / Issledovanie paratirina v syvorotke krovi u bol'nykh rakom vestibuliarnogo otdela gortani.

Measurements of parathyroid hormone concentrations were made in the serum of patients with vestibular larynx before treatment, after radiation treatment and after radiation treatment followed by surgery. Patients with laryngeal cancer were found to have impaired calcium metabolism, their serum parathyroid hormone concentrations were elevated. After radiotherapy, in a favorable clinical course, patients with cancer of the vestibular larynx stage II show normalization of the hormone levels, whereas the above combined treatment diminished the concentrations, but they still remain above normal.

Role of the ADP/ATP and aspartate/glutamate antiporters in the uncoupling effect of fatty acids, lauryl sulfate, and 2, 4-dinitrophenol in liver mitochondria.

Study of the uncoupling effect of various saturated fatty acids (from caprylic to palmitic) revealed that the glutamate recoupling effect was more pronounced in the case of short chain fatty acids, whereas recoupling of mitochondria by carboxyatractylate was more effective in the case of long chain fatty acids. The overall recoupling effect, however, did not depend on the fatty acid chain length. Besides carboxyatractylate, glutamate and aspartate also exhibited a recoupling effect under uncoupling by lauryl sulfate. The uncoupling effect of lauryl sulfate was markedly weaker in the presence of DNP or laurate (but not FCCP) which were added in concentrations causing twofold increase in mitochondrial respiration. In the presence of lauryl sulfate the uncoupling action of laurate and DNP was insensitive to carboxyatractylate and glutamate. With laurate and DNP as uncouplers increasing the pH from 7.0 to 7.8 potentiated the recoupling effect of carboxyatractylate and attenuated the recoupling effect of glutamate. In the case of uncoupling by lauryl sulfate similar changes in the recoupling effect of carboxyatractylate and glutamate were observed only in the presence of 10 microM tetraphenylphosphonium. Thus, when uncoupling is induced by fatty acids, DNP, and lauryl sulfate, the ADP/ATP and aspartate/glutamate antiporters function as two parallel and independent pathways for mitochondrial membrane potential dissipation. We suggest that the role of the ADP/ATP antiporter in uncoupling includes proton capture from the intermembrane space with subsequent protonation of uncoupler anions, their transport as neutral molecules on the internal side, and deprotonation followed by proton release into the matrix and transfer of the uncoupler anion in the reverse direction. During uncoupling the aspartate/glutamate antiporter cyclically carries the uncoupler anion with simultaneous proton transfer from the intermembrane space into the matrix.

The anion-carrier mediated uncoupling effect of dicarboxylic fatty acids in liver mitochondria depends on the position of the second carboxyl group.

Effects of dicarboxylic fatty acids with varying positions of the carboxyl groups on respiration and membrane potential of liver mitochondria were studied. Tetradecylmalonic acid (a fatty acid with two carboxyl groups in the alpha-position) efficiently uncoupled oxidative phosphorylation similarly to palmitic acid with the same number of carbon atoms. Similarly to the uncoupling by palmitic acid, the coupling effects of carboxyatractylate and glutamate changed reciprocally with changes in pH of the incubation medium: on increasing the pH from 7.0 to 7.8, the coupling effect of carboxyatractylate increased and that of glutamate decreased. A dicarboxylic fatty acid with the second carboxyl at the end of the alkyl chain in the omega-position (alpha, omega-tetradecyldicarboxylic acid) stimulated respiration of the mitochondria at a significantly higher concentration than myristic acid with the same number of carbon atoms, but unlike the latter nearly failed to decrease the transmembrane potential DeltaPsi. Neither carboxyatractylate nor glutamate inhibited the respiration stimulated by this dicarboxylic fatty acid.

[Treatment-related aldosterone changes in cancer of the middle part of the larynx]. / Issledovanie dinamiki al'dosterona pri rake srednego otdela gortani v zavisimosti ot vida provedennoi terapii.

Radioimmunoassay was employed to investigate time course changes in blood serum aldosteron in 28 patients with cancer of the middle laryngeal part before and after radiation treatment as well as after radiation treatment followed by surgical intervention. Initial values of aldosteron were low evidencing impaired mineral-corticoid activity of the adrenal cortex. Aldosteron returned to normal after effective radiotherapy at early tumor stage and after radical surgical treatment at later cancer stages. Hormonal unbalance provides more significant information on tumor involvement of the larynx and is a good indicator of the treatment efficiency.

Involvement of aspartate/glutamate antiporter in fatty acid-induced uncoupling of liver mitochondria.

Effects of aspartate, glutamate and an inhibitor of the aspartate/glutamate antiporter, diethylpyrocarbonate (DEPC), on uncoupling of the energy transduction processes in rat liver mitochondria have been investigated. It is found that both the antiporter substrates and the antiporter inhibitor operate as recouplers when uncoupling is caused by free fatty acids (FFA). Recoupling consists in (1) partial inhibition of the FFA-stimulated respiration and (2) some increase in the membrane potential. Half-maximal effects are observed at concentrations of glutamate and aspartate close the K(m) values of the antiporter. Recouplings by glutamate (aspartate) and DEPC are not additive. On the other hand, recoupling by any of these compounds and carboxyatractylate or ADP appears to be additive. Uncoupling by dinitrophenol is less sensitive to the recouplers whereas that by FCCP is not sensitive at all. It is concluded that uncoupling by FFA in rat liver mitochondria is mediated not only by the ATP/ADP antiporter but also by the aspartate/glutamate antiporter.

[The changes of serum cortisol and aldosterone in patients with cancer of vestibular section of larynx after irradiation and with and without surgery]. / Dinamika kortizola i al'dosterona v syvorotke krovi bol'nykh rakom vestibuliarnogo otdela gortani posle luchevogo lecheniia s posleduiushchim khirurgicheskim vmeshatel'stvom i bez nego.

Enzyme immunoassay was used to measure serum levels of hydrocortisone and aldosterone in 85 patients with vestibular laryngeal cancer before treatment and after it. The treatment consisted of either radiation alone or radiation followed by surgery. Pretreatment examination revealed adrenal dysfunction evident from enhanced glucocorticoid and inhibited mineral corticoid activity. Radiotherapy alone or in combination with surgery gave rise to a trend to a decline in glucocorticoid activity and stimulated mineral corticoid activity.

Carboxyatractylate- and cyclosporin A- sensitive uncoupling in liver mitochondria of ground squirrels during hibernation and arousal.

Oxygen consumption and transmembrane electrical potential difference in liver mitochondria from hibernating, arousing and active ground squirrels have been compared. It is found that arousal from hibernation is accompanied by uncoupling which is mediated by two different mechanisms, one sensitive to cyclosporin A and the other suppressed by carboxyatractylate. Both uncoupling effects reach their maxima at 20 - 25 degrees C body temperatures. Involvement of an increase in the free fatty acid concentration in the arousal-induced uncoupling mechanisms is discussed.

[Participation of the ADP/ATP-antiporter in the uncoupling action of fatty acids in liver mitochondria]. / Uchastie ATP/ADP-antiportera v razobshchaiushchem deistvii zhirnykh kislot v mitokhondriiakh pecheni.

The controversial data on the involvement of the ATP/ADP-antiporter in the uncoupling effect of fatty acids in liver mitochondria as well as on the sensitivity of the uncoupling process to the ATP/ADP-antiporter inhibitor, carboxyatractylate, have been analyzed. It has been shown that in liver mitochondria uncoupled by palmitic acid, pyridoxal-5-phosphate, diethyl pyrocarbonate and glutamate produce an additional recoupling action against the background of carboxyatractylate. No such effect is observed during mitochondrial uncoupling by FCCP. Micromolar concentrations of ADP added prior to palmitic acid produce a far more potent coupling action; the subsequent recoupling effect of carboxyatractylate diminishes thereby. This finding suggests that the reduction of endogenous ADP is responsible for the increased sensitivity of the uncoupling action of fatty acids to carboxyatractylate. It is concluded that the ATP/ADP-antiporter and other membrane carriers play a role in the uncoupling action of fatty acids on liver mitochondria.

Fatty acid-induced Ca(2+)-dependent uncoupling and activation of external pathway of NADH oxidation are coupled to cyclosporin A-sensitive mitochondrial permeability transition.

Permeabilization of inner mitochondrial membrane by palmitic acid in the presence of Ca2+ (cyclosporin A-sensitive stimulation of respiration, decrease of delta psi and high amplitude swelling) is accompanied by activation of the external pathway of NADH oxidation in liver mitochondria. The "pore"-sealing agents (cyclosporin A, Mg2+ with ADP, and L-carnitine with ATP) are equally effective in preventing the induction of external pathway of NADH oxidation by Ca2+ with palmitate. However, activities of these agents are different in respect to recoupling of permeabilized mitochondria. Participation of cyclosporin A-sensitive "pore" in the fatty acid- and Ca(2+)-dependent induction of external pathway of NADH oxidation and in Ca(2+)-dependent uncoupling is discussed.