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Biomedical subjects

Giorgio Lenaz

Publications and source records attributed to Giorgio Lenaz.

At least 19 recordsLinked to original sources
PubMed2005

Potential antitumor agents. 37. Synthesis and antitumor activity of guanylhydrazones from imidazo[2,1-b]thiazoles and from the new heterocyclic system thiazolo[2',3':2,3]imidazo[4,5-c]quinoline.

This paper reports synthesis and antitumor activity of new guanylhydrazones from imidazo[2,1-b]thiazoles and from the new heterocyclic system thiazolo[2',3':2,3]imidazo[4,5-c]quinoline. The compounds were tested as potential antitumor agents at the National Cancer Institute. The effect of the guanylhydrazone of 2-chloro-6-(2,5-dimethoxy-4-nitrophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde (41) was investigated, and it was found to be an inhibitor of Complex III of the mitochondrial respiratory chain and is able to induce apoptosis in the cell lines HT29 and HL60.

Animals↗
PubMed2005

Severe impairment of complex I-driven adenosine triphosphate synthesis in leber hereditary optic neuropathy cybrids.

BACKGROUND: Leber hereditary optic neuropathy (LHON) is a maternally inherited form of central vision loss associated with mitochondrial DNA point mutations that affect the ND subunits of complex I. OBJECTIVE: To elucidate the bioenergetic consequences of complex I dysfunction in LHON. DESIGN: The biochemical phenotypes of LHON mutations have been investigated using the transmitochondrial cytoplasmic hybrid (cybrid) cell model derived from the osteocarcoma parental cell line 143B.TK-. SETTING: Research laboratories at neuroscience and biochemistry departments at the University of Bologna, Scientific Institute "E. Medea," and University of College Medical School. PARTICIPANTS: Fibroblast cell lines were obtained from patients affected with LHON, as defined by the presence of 1 pathogenic mutation, and from healthy volunteers as controls to construct cybrid cell lines. MAIN OUTCOME MEASURES: Complex I (glutamate-malate)- and complex II (succinate)-dependent adenosine triphosphate (ATP) synthesis, their respective respiratory rates, and total cellular ATP content were investigated using digitonin permeabilized cybrid cells. Multiple cybrid cell lines were constructed, introducing into osteosarcoma-derived rho(0) cells either wild-type or LHON mutant mitochondria carrying each of the 3 common mutations at positions 11778/ND4, 3460/ND1, and 14484/ND6. RESULTS: All 3 LHON mutations impaired ATP synthesis and the respiratory control ratio driven by complex I substrates. In contrast, succinate-driven ATP synthesis, respiration rates, and respiratory control ratios were not affected. However, the defective ATP synthesis with complex I substrates did not result in reduced ATP cellular content, indicating a compensatory mechanism. CONCLUSIONS: The LHON pathogenic mutations profoundly impair complex I-dependent synthesis of ATP, providing a common biochemical feature that may play a major role in LHON pathogenesis. Stratification of the results by mutation suggests that the 11778/ND4 mutation may induce an uncoupling of cybrid respiration, whereas the other 2 mutations impair the oxygen consumption rate.

Adenosine Triphosphate↗
PubMed2004

Biochemical analysis of respiratory function in cybrid cell lines harbouring mitochondrial DNA mutations.

We analysed key biochemical features that reflect the balance between glycolysis and glucose oxidation in cybrids (cytoplasmic hybrids) harbouring a representative sample of mitochondrial DNA point mutations and deletions. The cybrids analysed had the same 143B cell nuclear background and were isogenic for the mitochondrial background. The 143B cell line and its rho(0) counterpart were used as controls. All cells analysed were in a dynamic state, and cell number, time of plating, culture medium, extracellular volume and time of harvest and assay were strictly controlled. Intra- and extra-cellular lactate and pyruvate levels were measured in homoplasmic wild-type and mutant cells, and correlated with rates of ATP synthesis and O2 consumption. In all mutant cell lines, except those with the T8993C mutation in the ATPase 6 gene, glycolysis was increased even under conditions of low glucose, as demonstrated by increased levels of extracellular lactate and pyruvate. Extracellular lactate levels were strictly and inversely correlated with rates of ATP synthesis and O2 consumption. These results show increased glycolysis and defective oxidative phosphorylation, irrespective of the type or site of the point mutation or deletion in the mitochondrial genome. The different biochemical consequences of the T8993C mutation suggest a uniquely different pathogenic mechanism for this mutation. However, the distinct clinical features associated with some of these mutations still remain to be elucidated.

Adenosine Triphosphate↗
PubMed2004

Effects of new ubiquinone-imidazo[2,1-b]thiazoles on mitochondrial complex I (NADH-ubiquinone reductase) and on mitochondrial permeability transition pore.

In this work we describe the synthesis of a series of imidazo[2,1-b]thiazoles and 2,3-dihydroimidazo[2,1-b]thiazoles connected by means of a methylene bridge to CoQ(0). These compounds were tested as specific inhibitors of the NADH:ubiquinone reductase activity in mitochondrial membranes. The imidazothiazole system when bound to the quinone ring in place of the isoprenoid lateral side chain, may increase the inhibitory effect (with an IC(50) for NADH-Q(1) activity ranging between 0.25 and 0.96 microM) whereas the benzoquinone moiety seems to lose the capability to accept electrons from complex I as indicated by very low maximal velocity elicited by the compounds tested. Moreover the low rotenone sensitivity for almost all of these compounds suggests that they are only partially able to interact with the physiological ubiquinone-reduction site. The compounds were investigated for the capability of increasing the permeability transition of the inner mitochondrial membrane in isolated mitochondria. Unlike CoQ(0), which is considered a mitochondrial membrane permeability transition inhibitor, the new compounds were inducers.

Animals↗
PubMed2004

Heterologous mitochondrial DNA recombination in human cells.

Inter-molecular heterologous mitochondrial DNA (mtDNA) recombination is known to occur in yeast and plants. Nevertheless, its occurrence in human cells is still controversial. To address this issue we have fused two human cytoplasmic hybrid cell lines, each containing a distinct pathogenic mtDNA mutation and specific sets of genetic markers. In this hybrid model, we found direct evidence of recombination between these two mtDNA haplotypes. Recombinant mtDNA molecules in the hybrid cells were identified using three independent experimental approaches. First, recombinant molecules containing genetic markers from both parental alleles were demonstrated with restriction fragment length polymorphism of polymerase chain reaction products, by measuring the relative frequencies of each marker. Second, fragments of recombinant mtDNA were cloned and sequenced to identify the regions involved in the recombination events. Finally, recombinant molecules were demonstrated directly by Southern blot using appropriate combinations of polymorphic restriction sites and probes. This combined approach confirmed the existence of heterogeneous species of recombinant mtDNA molecules in the hybrid cells. These findings have important implications for mtDNA-related diseases, the interpretation of human evolution and population genetics and forensic analyses based on mtDNA genotyping.

Blotting, Southern↗
PubMed2004

Bioenergetics of mitochondrial diseases associated with mtDNA mutations.

This mini-review summarizes our present view of the biochemical alterations associated with mitochondrial DNA (mtDNA) point mutations. Mitochondrial cytopathies caused by mutations of mtDNA are well-known genetic and clinical entities, but the biochemical pathogenic mechanisms are often obscure. Leber's hereditary optic neuropathy (LHON) is due to three main mutations in genes for complex I subunits. Even if the catalytic activity of complex I is maintained except in cells carrying the 3460/ND1 mutation, in all cases there is a change in sensitivity to complex I inhibitors and an impairment of mitochondrial respiration, eliciting the possibility of generation of reactive oxygen species (ROS) by the complex. Neurogenic muscle weakness, Ataxia and Retinitis Pigmentosa (NARP), is due to a mutation in the ATPase-6 gene. In NARP patients ATP synthesis is strongly depressed to an extent proportional to the mutation load; nevertheless, ATP hydrolysis and ATP-driven proton translocation are not affected. It is suggested that the NARP mutation affects the ability of the enzyme to couple proton transport to ATP synthesis. A point mutation in subunit III of cytochrome c oxidase is accompanied by a syndrome resembling MELAS: however, no major biochemical defect is found, if we except an enhanced production of ROS. The mechanism of such enhancement is at present unknown. In this review, we draw attention to a few examples in which the overproduction of ROS might represent a common step in the induction of clinical phenotypes and/or in the progression of several human pathologies associated with mtDNA point mutations.

Adenosine Triphosphatases↗
PubMed2004

Bioenergetics shapes cellular death pathways in Leber's hereditary optic neuropathy: a model of mitochondrial neurodegeneration.

Leber's hereditary optic neuropathy (LHON) was the first maternally inherited disease to be associated with point mutations in mitochondrial DNA and is now considered the most prevalent mitochondrial disorder. The pathology is characterized by selective loss of ganglion cells in the retina leading to central vision loss and optic atrophy, prevalently in young males. The pathogenic mtDNA point mutations for LHON affect complex I with the double effect of lowering the ATP synthesis driven by complex I substrates and increasing oxidative stress chronically. In this review, we first consider the biochemical changes associated with the proton-translocating NADH-quinone oxidoreductase of mitochondria in cybrid cells carrying the most common LHON mutations. However, the LHON cybrid bioenergetic dysfunction is essentially compensated under normal conditions, i.e. in glucose medium, but is unrevealed by stressful conditions such as growing cybrids in glucose free/galactose medium, which forces cells to rely only on respiratory chain for ATP synthesis. In fact, the second part of this review deals with the investigation of LHON cybrid death pathway in galactose medium. The parallel marked changes in antioxidant enzymes, during the time-course of galactose experiments, also reveal a relevant role played by oxidative stress. The LHON cybrid model sheds light on the complex interplay amongst the different levels of biochemical consequences deriving from complex I mutations in determining neurodegeneration in LHON, and suggests an unsuspected role of bioenergetics in shaping cell death pathways.

Animals↗
PubMed2004

The mitochondrial respiratory chain is partially organized in a supercomplex assembly: kinetic evidence using flux control analysis.

The model of the respiratory chain in which the enzyme complexes are independently embedded in the lipid bilayer of the inner mitochondrial membrane and connected by randomly diffusing coenzyme Q and cytochrome c is mostly favored. However, multicomplex units can be isolated from mammalian mitochondria, suggesting a model based on direct electron channeling between complexes. Kinetic testing using metabolic flux control analysis can discriminate between the two models: the former model implies that each enzyme may be rate-controlling to a different extent, whereas in the latter, the whole metabolic pathway would behave as a single supercomplex and inhibition of any one of its components would elicit the same flux control. In particular, in the absence of other components of the oxidative phosphorylation apparatus (i.e. ATP synthase, membrane potential, carriers), the existence of a supercomplex would elicit a flux control coefficient near unity for each respiratory complex, and the sum of all coefficients would be well above unity. Using bovine heart mitochondria and submitochondrial particles devoid of substrate permeability barriers, we investigated the flux control coefficients of the complexes involved in aerobic NADH oxidation (I, III, IV) and in succinate oxidation (II, III, IV). Both Complexes I and III were found to be highly rate-controlling over NADH oxidation, a strong kinetic evidence suggesting the existence of functionally relevant association between the two complexes, whereas Complex IV appears randomly distributed. Moreover, we show that Complex II is fully rate-limiting for succinate oxidation, clearly indicating the absence of substrate channeling toward Complexes III and IV.

Adenosine Triphosphate↗
PubMed2004

Yessotoxin, a shellfish biotoxin, is a potent inducer of the permeability transition in isolated mitochondria and intact cells.

The diarrhetic poisoning by bivalve molluscs, diarrhetic shellfish poisoning, is due to consumption of mussels containing biotoxins produced by some Dinoflagellate species. Toxic effects of yessotoxin (YTX) include morphological alterations of mitochondria from heart and liver but the biochemical basis for these alterations is completely unknown. This paper demonstrates that YTX is a very powerful compound that opens the permeability transition pore (PTP) of the inner mitochondrial membrane of rat liver mitochondria at nanomolar concentrations. The effect requires the presence of a permissive level of calcium, by itself incapable of opening the pore. The direct effect of YTX on PTP is further confirmed by the inhibition exerted by cyclosporin A (CsA) that is known as a powerful inhibitor of PTP opening. Moreover, YTX induces membrane depolarization as shown by the quenching of tetramethylrhodamine methyl ester (TMRM), also prevented by the addition of CsA. YTX caused PTP opening in Morris Hepatoma 1C1 cells, as shown by the occurrence of CsA-sensitive depolarization within minutes of the addition of submicromolar concentrations of the toxin. These results provide a biochemical basis for the mitochondrial alterations observed in the course of intoxication with YTX, offering the first clue into the pathogenesis of diseases caused by YTX, and providing a novel tool to study the PTP in situ.

Animals↗
PubMed2004

The mtDNA T8993G (NARP) mutation results in an impairment of oxidative phosphorylation that can be improved by antioxidants.

A T8993G point mutation in the mtDNA results in a Leu156Arg substitution in the MTATP6 subunit of the mitochondrial F1F0-ATPase. The T8993G mutation causes impaired oxidative phosphorylation (OXPHOS) in two mitochondrial disorders, NARP (neuropathy, ataxia and retinitis pigmentosa) and MILS (maternally inherited Leigh's syndrome). It has been reported, in some studies, that the T8993G mutation results in loss of assembled F1F0-ATPase. Others reported that the mutation causes impairment of proton flow through F0. In addition, it was shown that fibroblasts from NARP subjects have a tendency to undergo apoptotic cell death, perhaps as a result of increased free radical production. Here, we show that the T8993G mutation inhibits oxidative phosphorylation and results in enhanced free radical production. We suggest that free radical-mediated inhibition of OXPHOS contributes to the loss of ATP synthesis. Importantly, we show that antioxidants restore respiration and partially rescue ATP synthesis in cells harboring the T8993G mutation. Our results indicate that free radicals might play an important role in the pathogenesis of NARP/MILS and that this can be prevented by antioxidants. The effectiveness of antioxidant agents in cultured NARP/MILS cells suggests that they might have a potential beneficial role in the treatment of patients with NARP.

Adenosine Triphosphate↗
PubMed2004

Increased transcription of mitochondrial genes for Complex I in human platelets during ageing.

We studied the effect of ageing on the mRNA levels of mitochondria-encoded polypeptides in human platelets. We used quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) to investigate the expression of selected cytochrome c oxidase (COX) genes (subunits I and III) and Complex I genes (subunits reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ND)1 and (ND)5 in platelets from young and aged healthy subjects. Northern blot analysis confirmed the PCR results. COX I expression is higher than that of COX III in both young and aged platelets. A significant increase of transcripts for Complex I was found during ageing. On the contrary, the mRNA levels of the two COX subunits did not significantly vary during ageing.

Adult↗
PubMed2004

Plasma membrane oxidoreductase activity in cultured cells in relation to mitochondrial function and oxidative stress.

Dichlorophenol indophenol (DCIP) reduction by intracellualr pyridine nucleotides was investigated in two different lines of cultured cells characterized by enhanced production of reacive oxygen species (ROS) with respect to suitable controls. The first line denominated XTC-UC1 was derived from a metastasis of an oxyphilic thyroid tumor characterized by mitochondrial hyperplasia and compared with a line (B-CPAP) derived from a papillary thyroid carcinoma with normal mitochondrial mass. The second line (170 MN) was a cybrid line derived from rho0 cells from an osteosarcoma line (143B) fused with platelets from a patient with a nucleotide 9957 mutation in mitochondrial DNA (encoding for cytochrome c oxidase subunit III) in comparison with the parent 143B line. The experimental lines had no major decreases of electron transfer activities with respect to the controls; both of them, however, exhibited an increased peroxide production. The XTC-UC1 cell line exhibited enhanced activity with respect to control of dicoumarol-sensitive DCIP reduction, identified with membrane bound DT-diaphorase, whereas dicoumarol insensitive DCIP reduction was not significantly changed. On the other hand the mtDNA mutated cybrids exhibited a strong increase of both dicoumarol sensitive and insensitive DCIP reduction. The results suggest that enhanced oxidative stress and not deficient respiratory activity per se is the stimulus triggering over-expression of plasma membrane oxidative enzymes.

Breast Neoplasms↗
PubMed2004

The mitochondrial production of reactive oxygen species in relation to aging and pathology.

Mitochondria are known to be strong producers of reactive oxygen species (ROS) and, at the same time, particularly susceptible to the oxidative damage produced by their action on lipids, proteins, and DNA. In particular, damage to mtDNA induces alterations to the polypeptides encoded by mtDNA in the respiratory complexes, with consequent decrease of electron transfer, leading to further production of ROS and thus establishing a vicious circle of oxidative stress and energetic decline. This deficiency in mitochondrial energetic capacity is considered the cause of aging and age-related degenerative diseases. Complex I would be the enzyme most affected by ROS, since it contains seven of the 13 subunits encoded by mtDNA. Accordingly, we found that complex I activity is significantly affected by aging in rat brain and liver mitochondria as well as in human platelets. Moreover, due to its rate control over aerobic respiration, such alterations are reflected on the entire oxidative phosphorylation system. We also investigated the role of mitochondrial complex I in superoxide production and found that the one-electron donor to oxygen is most probably the Fe-S cluster N2. Short chain coenzyme Q (CoQ) analogues enhance ROS formation, presumably by mediating electron transfer from N2 to oxygen, both in bovine heart SMP and in cultured HL60 cells. Nevertheless, we have accumulated much evidence of the antioxidant role of reduced CoQ(10) in several cellular systems and demonstrated the importance of DT-diaphorase and other internal cellular reductases to reduce exogenous CoQ(10) after incorporation.

Aging↗
PubMed2004

Methods to detect mitochondrial function.

The bioenergetic function of mitochondria can be investigated in intact cells by a variety of methods. A simple biochemical method to compare mitochondrial oxidative phosphorylation with glycolytic ATP synthesis takes advantage of the Pasteur effect, since the amount of lactate produced under basal conditions is an indication of glycolytic ATP, while the Delta-lactate (the difference between excess lactate produced after inhibition of respiration and basal lactate) represents ATP produced anaerobically in order to compensate for decreased oxidative phosphorylation after respiratory chain inhibition. The system has been validated in a series of cells, including human platelets and lymphocytes and lines cultured in vitro. Measurement of KCN-sensitive oxygen consumption by the cells and its sensitivity to uncouplers can be good supplementary indication of mitochondrial phosphorylative capacity.

Adenosine Triphosphate↗
PubMed2004

Impairment of mitochondrial oxidative phosphorylation in rat fatty liver exposed to preservation-reperfusion injury.

BACKGROUND/AIMS: As the impairment of the cellular energy metabolism contributes to the failure of fatty liver grafts after transplantation, we aimed to determine whether steatosis affects the oxidative phosphorylation activity during preservation. METHODS: Rat normal and fatty livers were preserved for 18 h and then reperfused with warm oxygenated solution. The oxidative phosphorylation, the F(0)F(1)-ATPase and the Complex I activities were assessed in isolated mitochondria before and after preservation, and during reperfusion. The ALT release and portal pressure were monitored during reperfusion. RESULTS: The baseline phosphorylation activity was similar in normal and steatotic mitochondria. After cold preservation, the respiratory control index and state 3 respiration decreased significantly only in steatotic livers. Reperfusion induced a further deterioration in either group. Contrary to normal liver, uncoupling of fatty liver mitochondria allowed the recovery of the maximal respiration rate only using succinate (Complex II-dependent substrate), but not glutamate-malate (Complex I-dependent). Complex I dysfunction was confirmed spectrophotometrically. The ATPase activity was also significantly lower in fatty livers. Finally, ALT release and portal pressure were greater in steatotic livers. CONCLUSIONS: The alteration of the oxidative phosphorylation activity during preservation is greatly exacerbated by fatty infiltration likely resulting from damage of the respiratory chain Complex I and of the F(0)F(1)-ATP synthase.

Adenosine Triphosphate↗
PubMed2004

Evaluation of cellular energetics by the pasteur effect in intact cardiomyoblasts and isolated perfused hearts.

This work aims at exploring changes in cellular energetics by exploiting the Pasteur effect. We assumed that lactate overproduction arising from antimycin A-induced inhibition of mitochondrial respiration (delta-lactate = stimulated [lactate] -basal [lactate]) is indicative of the energy provided aerobically by the cell. Rat embryonal cardiomyocytes (H9c2), incubated with 2 micromol/L antimycin A, increased about 6 fold their lactate production in a manner linear with time and cell number. Antimycin A was also delivered to Langendorff-perfused rat hearts under control aerobic conditions or after 20 min-ischemia and 30 min-reperfusion. The test started at the end of each perfusion and lactate was measured into perfusate collected for further 25 min. A cardioplegic solution was also delivered during the test to exclude that lactate production was influenced by cardiac contraction. Control delta-lactate was 20.9 +/- 2.31 (S.E.M.) microg/mL and markedly decreased after reperfusion (7.66 +/- 0.51, p < 0.001), showing that energy production was impaired of about 70%. The determination of oxygen consumption by mitochondria isolated from reperfused hearts also suggested that the damage to the respiratory chain was similar to that evaluated by lactate overproduction (Respiratory Control Index: 75% lower than control, p < 0.001). Moreover, when delta-lactate was referred to the estimated cells which remained viable at the end of reperfusion (49.9%), it was 25% lower than control (p < 0.05). Therefore, we proposed this test as a tool for quantifying both physiological and pathological energetic modifications in living intact cardiomyocytes and in isolated and perfused hearts.

Animals↗
PubMed2004

Oxidation of glycerol-3-phosphate in porcine and bovineadrenal cortex mitochondria.

The capabilities of porcine adrenal cortex mitochondria to oxidize glycerol-3-phosphate (GP) were studied. In comparison with bovine adrenal cortex mitochondria, porcine mitochondria oxidized GP about three times more actively (18.9 vs 6.1 nmol O(2)/min per mg protein in the presence of ADP) and the activity of mitochondrial glycerol-3-phosphate dehydrogenase was about four times higher (33.4 vs 8.2 nmol/min per mg protein). In porcine adrenal cortex mitochondria we found similar values for succinate and GP oxidation both in the absence and presence of ADP or deoxycorticosterone (DOC). Rotenone sensitivity of DOC stimulation of GP oxidation indicated that porcine adrenal cortex mitochondria are able to oxidize GP and thus to generate NADPH from GP, presumably via reverse electron transport followed by energy-dependent NADH-NADP transhydrogenation.

Adrenal Cortex↗