Wilms' tumor protein and FLT3-internal tandem duplication expression in patients with de novo acute myeloid leukemia.

Bone marrow samples of 30 patients with de novo adult acute myeloid leukemia (AML) were analyzed for Wt1 and FLT3-internal tandem duplication (FLT3-ITD) expression measured by western blot and reverse transcription-polymerase chain reaction analysis, respectively. Wt1 was detected in 53·3% of AML patients (16/30), while FLT3-ITD in 23·3% (7/30). The high Wt1 expression correlated with the presence of FLT3-ITD (P = 0·014) and lower rate of complete remission (P = 0·023). The cumulative survival in AML patients was affected significantly by the presence of FLT3-ITD, being lower in the FLT3-ITD (+) group (6·0±2·4 months) compared to the FLT3-ITD (-) patients (17·9±3·3; P = 0·04). The expression of FLT3-ITD could probably activate Wt1 expression in AML blast cells and thus might contribute to its oncogenic function to provide cells with survival advantages in vivo. The detection of both molecular markers (Wt1 and/or FLT3-ITD) may be helpful in defining high risk AML patients that need special therapeutic strategies.

Is supercomplex organization of the respiratory chain required for optimal electron transfer activity?

The supra-molecular assembly of the main respiratory chain enzymatic complexes in the form of "super-complexes" has been proved by structural and functional experimental evidence. This evidence strongly contrasts the previously accepted Random Diffusion Model stating that the complexes are functionally connected by lateral diffusion of small redox molecules (i.e. Coenzyme Q and cytochrome c). This review critically examines the available evidence and provides an analysis of the functional consequences of the intermolecular association of the respiratory complexes pointing out the role of Coenzyme Q and of cytochrome c as channeled or as freely diffusing intermediates in the electron transfer activity of their partner enzymes.

Control of respiration by nitric oxide in Keilin-Hartree particles, mitochondria and SH-SY5Y neuroblastoma cells.

The pattern of cytochrome c oxidase inhibition by nitric oxide (NO) was investigated polarographically using Keilin-Hartree particles, mitochondria and human neuroblastoma cells. NO reacts with purified cytochrome c oxidase forming either a nitrosyl- or a nitrite-inhibited derivative, displaying distinct kinetics and light sensitivity of respiration recovery in the absence of free NO. Keilin-Hartree particles or cells, respiring either on endogenous substrates alone or in the presence of ascorbate, as well as state 3 and state 4 mitochondria respiring on glutamate and malate, displayed the rapid recovery characteristic of the nitrite derivative. All systems, when respiring in the presence of tetramethyl-p-phenylenediamine, were characterised by the slower, light-sensitive recovery typical of the nitrosyl derivative. Together the results suggest that the reaction of NO with cytochrome c oxidase in situ follows two alternative inhibition pathways, depending on the electron flux through the respiratory chain.

The site of production of superoxide radical in mitochondrial Complex I is not a bound ubisemiquinone but presumably iron-sulfur cluster N2.

The mitochondrial respiratory chain is a powerful source of reactive oxygen species, considered as the pathogenic agent of many diseases and of aging. We have investigated the role of Complex I in superoxide radical production in bovine heart submitochondrial particles and found, by combined use of specific inhibitors of Complex I and by Coenzyme Q (CoQ) extraction from the particles, that the one-electron donor in the Complex to oxygen is a redox center located prior to the binding sites of three different types of CoQ antagonists, to be identified with a Fe-S cluster, most probably N2 on the basis of several known properties of this cluster. Short chain CoQ analogs enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analog, idebenone, is particularly effective in promoting superoxide formation.

Does coenzyme Q10 play a role in opposing oxidative stress in patients with age-related macular degeneration?

To seek some specific biochemical markers of age-related macular degeneration (AMD), coenzyme Q10 (CoQ10) levels were determined in plasma and platelets from 19 exudative AMD patients and 19 age-matched controls. Lipid peroxidation was followed in plasma in vitro after the addition of a free radical initiator. Most patients had lower plasma CoQ10 content than most controls. Plasma from controls showed greater capacity to oppose the oxidative damage. These results support the concept that free radicals play a pathogenic role in AMD and that CoQ10 may have a protective effect.

Mitochondrial bioenergetics in aging.

Mitochondria are strongly involved in the production of reactive oxygen species, considered as the pathogenic agent of many diseases and of aging. The mitochondrial theory of aging considers somatic mutations of mitochondrial DNA induced by oxygen radicals as the primary cause of energy decline; experimentally, complex I appears to be mostly affected and to become strongly rate limiting for electron transfer. Mitochondrial bioenergetics is also deranged in human platelets upon aging, as shown by the decreased Pasteur effect (enhancement of lactate production by respiratory chain inhibition). Cells counteract oxidative stress by antioxidants; among lipophilic antioxidants, coenzyme Q is the only one of endogenous biosynthesis. Exogenous coenzyme Q, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases.

Localization and mobility of coenzyme Q in lipid bilayers and membranes.

We have studied the mobility of coenzyme Q (CoQ) in lipid bilayers and mitochondrial membranes in relation to the control of electron transfer activities. A molecular dynamics computer simulation in the vacuum yielded a folded structure for CoQ10, with a length of only 21 A. Using this information we were able to calculate diffusion coefficients in the range of 10(-6) cm2/s in good agreement with those found experimentally by fluorescence quenching of pyrene derivatives. To investigate if CoQ diffusion may represent the rate-limiting step of electron transfer, we reconstituted complexes I and III and assayed the resulting NADH-cytochrome c reductase activity in presence of different CoQ10 levels and at different distances between complexes; the experimental turnovers were higher than the collision frequencies calculated using diffusion coefficients of 10(-9) cm2/s but compatible with values found by us by fluorescence quenching. Since the experimental turnovers are independent of the distance between complexes, we conclude that CoQ diffusion is not rate-limiting for electron transfer.

Protective effect of exogenous coenzyme Q in rats subjected to partial hepatic ischemia and reperfusion.

In a surgical model of liver ischemia lipid peroxidation occurs, as shown by increase of lipid peroxidation end products, endogenous CoQ9 is oxidized and mitochondrial respiration is lowered; however, pre-treatment of the rats by i.p. injection of CoQ10 for 14 days normalizes the above parameters, presumably by way of the observed high extent of reduction of the incorporated quinone; moreover, liver homogenates of the CoQ10-treated rats are more resistant than those of non-treated rats to oxidative stress induced by an azido free radical initiator. This preliminary study suggests that CoQ10 pre-treatment can be of beneficial effect against oxidative damage during liver surgery transplantation.

Histomorphometric studies in rat cerebral cortex: normal aging and cell loss.

One characteristic feature of the aged central nervous system (CNS) is neuron loss. Programmed cell death (PCD) has been implicated in neuronal death during development and may be involved in a number of age-related neurodegenerative diseases of the CNS. Cell death in the aging cerebral cortex was investigated in the present morphometric and immunohistochemical study of rat frontal cortex by detection of bcl-2 as the factor preventing PCD. The results were interpreted in the light of the bioenergetic features of aged motoneuron cells. Our results showed that 1) bcl-2 does not influence neuronal survival, and ii) the presence in aging frontal cortex of minor cellular morphometric and bioenergetic modifications, confirming the difference between normal aging and neurodegenerative disease.

Oxidative stress, antioxidant defences and aging.

Apoptosis and aging share common mechanisms in oxidative stress and mitochondrial involvement. Treatment of cultured neuroblastoma cells with a radical initiator induced apoptosis; raise in hydrogen peroxide and release of cytochrome c from mitochondria preceded collapse of mitochondrial potential and cell death. In rat hepatocytes treated with adriamycin incubation with exogenous Coenzyme Q10 counteracted the drug-induced increase of hydrogen peroxide and the fall of the mitochondrial potential, thus demonstrating the quinone antioxidant effect. Complex I activity and its rotenone sensitivity decreased in brain cortex non-synaptic mitochondria from old rats; a 5 kb mitochondrial DNA deletion was found only in the old rats. A similar behavior was found in human platelets from old individuals. The postulated energy decline was confirmed by the inhibitor sensitivities of platelet aggregation and lactate production. The lack of the 5 kb deletion in platelets throws doubts on mitochondrial DNA lesions as the only causes of mitochondrial dysfunction in aging.

Mitochondrial complex I defects in aging.

According to the 'mitochondrial theory of aging' it is expected that the activity of NADH Coenzyme Q reductase (Complex I) would be most severely affected among mitochondrial enzymes, since mitochondrial DNA encodes for 7 subunits of this enzyme. Being these subunits the site of binding of the acceptor substrate (Coenzyme Q) and of most inhibitors of the enzyme, it is also expected that subtle kinetic changes of quinone affinity and enzyme inhibition could develop in aging before an overall loss of activity would be observed. The overall activity of Complex I was decreased in several tissues from aged rats, nevertheless it was found that direct assay of Complex I using artificial quinone acceptors may underevaluate the enzyme activity. The most acceptable results could be obtained by applying the 'pool equation' to calculate Complex I activity from aerobic NADH oxidation; using this method it was found that the decrease in Complex I activity in mitochondria from old animals was greater than the activity calculated by direct assay of NADH Coenzyme Q reductase. A decrease of NADH oxidation and its rotenone sensitivity was observed in nonsynaptic mitochondria, but not in synaptic 'light' and 'heavy' mitochondria of brain cortex from aged rats. In a study of Complex I activity in human platelet membranes we found that the enzyme activity was unchanged but the titre for half-inhibition by rotenone was significantly increased in aged individuals and proposed this change as a suitable biomarker of aging and age-related diseases.

Decrease of rotenone inhibition is a sensitive parameter of complex I damage in brain non-synaptic mitochondria of aged rats.

We investigated NADH oxidation in non-synaptic and synaptic mitochondria from brain cortex of 4- and 24-month-old rats. The NADH oxidase activity was significantly lower in non-synaptic mitochondria from aged rats; we also found a significant decrease of sensitivity of NADH oxidation to the specific Complex I inhibitor, rotenone. Since the rotenone-binding site encompasses Complex I subunits encoded by mtDNA, these results are in accordance with the mitochondrial theory of aging, whereby somatic mtDNA mutations are at the basis of cellular senescence. Accordingly, a 5 kb deletion was detected only in the cortex of the aged animals.

Mitochondrial respiratory chain features after gamma-irradiation.

Radiation provokes damage to DNA but also to membrane and protein structure. Radiolysis is a tool used very often in the study of free radical biological effects and of scavenger molecules effectiveness. Nitroimidazoles have been demonstrated to enhance the radiation effects on biological structures. The studies we have performed on isolated mitochondria irradiated, with and without nitroimidazoles, at a radiation dose equal to LD90, indicate that this treatment is not able to affect the structural and functional features investigated (ubiquinone-10, fatty acids, respiratory cytochrome levels or membrane fluidity and respiratory enzymatic activities), suggesting that an involvement of such externally produced radicals on membrane damage is unlikely. Moreover it was ascertained that the mitochondrial redox activities do not take part into the intracellular nitroimidazole reduction.

Coenzyme Q changes in liver and plasma in the rat after partial hepatectomy.

The levels of coenzyme Q were determined in blood plasma and regenerating liver mitochondria of hepatectomized rats, using as controls either sham-operated or non-operated animals. Mitochondrial CoQ9 content increased in sham-operated rats, whereas it was significantly lower in hepatectomized with respect to non-operated animals. Plasma CoQ9 levels decreased dramatically in hepatectomized animals, but increased strongly in sham-operated in comparison with non-operated rats. The data suggest the possibility of a rate-limiting step in CoQ biosynthesis in hepatectomized animals.

Coenzyme Q depletion in rat plasma after partial hepatectomy.

Coenzyme Q content was monitored in blood plasma and regenerating liver mitochondria of hepatectomized rats, using as controls either sham-operated or non-operated animals. Mitochondrial CoQ9 content increased in sham-operated rats, whilst it was significantly lower in hepatectomized in comparison with non-operated animals at all considered times. On the other hand plasma CoQ9 levels dramatically decreased in hepatectomized animals, while strongly increased in sham-operated in comparison with non-operated rats. The quinone decrease in hepatectomized animals is likely to be due to the attainment of a rate-limiting step in CoQ biosynthesis.

Inhibitor sensitivity of respiratory complex I in human platelets: a possible biomarker of ageing.

NADH-Coenzyme Q reductase was assayed in platelet mitochondrial membranes obtained from 19 pools of two venous blood samples from female young (19-30 years) individuals and 18 pools from aged ones (66-107 years). The enzyme activities were not significantly changed in the two groups, but a decrease of sensitivity to the specific inhibitor, rotenone, occurred in a substantial number of aged individuals. The results are in agreement with the predictions of the mitochondrial theory of ageing and may be used to develop a sensitive biomarker of the ageing process.

Major changes in complex I activity in mitochondria from aged rats may not be detected by direct assay of NADH:coenzyme Q reductase.

We have investigated the respiratory activities and the concentrations of respiratory chain components of mitochondria isolated from the livers and hearts of two groups of rats aged 6 and 24 months respectively. In comparison with the adult controls (6 months), in aged rats there was a decline in total aerobic NADH oxidation in both tissues; only minor (non-significant) changes, however, were found in NADH:coenzyme Q reductase and cytochrome oxidase activities, and there was no change in ubiquinol-cytochrome c reductase activity. The coenzyme Q levels were slightly decreased in mitochondria from both organs of aged rats. The lowered NADH oxidase activity is not due to the slight decrease observed in the coenzyme Q levels, but is the result of decreased Complex I activity. Since the assay of NADH:coenzyme Q reductase requires quinone analogues, none of which can evoke its maximal turnover [Estornell, Fato, Pallotti and Lenaz (1993) FEBS Lett. 332, 127-131], its activity has been calculated indirectly by taking advantage of the relationship that exists between NADH oxidation and ubiquinol oxidation through the coenzyme Q pool. The results, expressed in this way, show a drastic loss of activity of Complex I in both the heart and the liver of aged animals in comparison with adult controls.

Underevaluation of complex I activity by the direct assay of NADH-coenzyme Q reductase in rat liver mitochondria.

We have shown that the rate of NADH-coenzyme Q reductase in rat liver mitochondria, assayed using the decyl-ubiquinone analog DB, is underevaluated, probably as a result of its low water solubility. In view of drawbacks encountered using other more soluble acceptors in this system, we demonstrate that the most reliable assay of the physiological rate of CoQ reduction by Complex I is the indirect calculation from the total rate of NADH oxidation and the rate of ubiquinol oxidation, using the pool equation of Kröger and Klingenberg [(1973) Eur. J. Biochem. 34, 358-368].

Coenzyme Q content in synaptic and non-synaptic mitochondria from different brain regions in the ageing rat.

We investigated the Coenzyme Q (CoQ) content of different mitochondrial fractions [free mitochondria (FM), synaptic heavy (HM) and light mitochondria (LM)] from three brain areas (cortex, striatum, hippocampus) of rats at different ages. In rats from 2 to 26 months of age, we observed only small differences in total CoQ content (CoQ9 + CoQ10). In FM and LM fractions, values are very similar and appear to be much higher than in HM fractions. The CoQ10/CoQ9 ratios are much higher in brain mitochondria than in other organs, suggesting possible modifications of CoQ biosynthetic pathways in brain; nevertheless they appear to remain constant during ageing. CoQ9 and CoQ10 contents slowly decrease reaching their minimum in rats of 18 months of age, then increase in the older ages. Considering ageing as partially driven by a summation of free radical-mediated processes, we can hypothesize that damage occurring to biological structures in the first half of life might be followed by induction phenomena tending to re-establish the primitive levels of antioxidant molecules.

Protective effect of exogenous coenzyme Q against damage by adriamycin in perfused rat liver.

We have investigated the effect of rat liver perfusion with adriamycin on mitochondrial activities. Although the perfusion treatment per se induces some decline of respiratory activities, adriamycin strongly potentiates this effect; moreover the coenzyme Q9 content of the mitochondrial membrane is significantly lowered by the antibiotic. Coaddition of coenzyme Q10 in the perfusate significantly protects the mitochondria, not only from loss of respiratory activities but also of the endogenous CoQ9 content. Exogenous CoQ10 fails to enhance respiratory activities in control rats, not treated with adriamycin, even though CoQ concentration has been proven not to be kinetically saturating in the respiratory chain under physiological conditions. Thus, the beneficial effect of CoQ10 in the perfusate does not appear to be the result of its role in the respiratory chain but is a consequence of its antioxidant action.