Mitochondrial Enzyme Complex Activities Research Articles

Behavioral, biochemical and cellular correlates in the protective effect of sertraline against transient global ischemia induced behavioral despair: Possible involvement of nitric oxide-cyclic guanosine monophosphate study pathway

Post-stroke depression (PSD) is one of the psychiatric complications after stroke. Present study was conducted to elucidate the protective effect of sertraline and possible involvement of nitric oxide mechanism against transient global ischemia induced behavioral despair. Bilateral common carotid artery occlusion was given twice for 5 min at 10 min interval followed by 96 h reperfusion. Ischemia reperfusion significantly increased immobility period and decreased resistance to lateral push as compared to sham-operated group. Ischemia reperfusion caused significant oxidative damage and mitochondrial enzyme complex (I–III) dysfunction as compared to sham group. Sertraline (5 and 10 mg/kg) treatment significantly reduced immobility period, increased resistance to lateral push, attenuated oxidative damage and restored mitochondrial enzyme complex activities as compared to ischemia group. l-Arginine (100 mg/kg) or sildenafil (5 mg/kg) pretreatment with sertraline (5 mg/kg) significantly reversed the protective effect of sertraline. However, l-NAME (10 mg/kg) or 7NI (10 mg/kg) pretreatment with sertraline (5 mg/kg) significantly potentiated their protective effect which were significant as compared to their effect alone. The present study shows that nitric oxide modulation is involved in the protective effect of sertraline.

Protective effect of rofecoxib and nimesulide against intra-striatal quinolinic acid-induced behavioral, oxidative stress and mitochondrial dysfunctions in rats

Role of cyclooxygenase (COX) enzyme has been well documented in both physiological and pathological conditions. COX-1 and COX-2 converts arachidonic acid into prostaglandins. Non-selective inhibition of COXs produces undesirable effects, whereas selective COX-2 inhibition produces protective effects in various inflammatory diseases. Recently, cyclooxygenase (COX) inhibitors have been implicated as a neuroprotectant in the treatment of various neurodegenerative diseases. Quinolinic acid is an endogenous excitotoxin that causes neurotoxicity in diverse areas of the brain and produces motor dysfunction. Present study is an attempt to investigate the possible role of COX inhibitors (selective COX-2 inhibitor and preferential COX-2 inhibitors) against quinolinic acid-induced behavioral, oxidative stress and mitochondrial enzyme complex alterations in rats. Intra-striatal administration of quinolinic acid (300 nmol) caused significant reduction in body weight (9%), motor in-coordination, oxidative damage [increased MDA (100%), nitrite concentration (195%), depleted SOD (71%), catalase levels (70%)] and alteration in mitochondrial enzyme complex activity (decreased complex I (50%), II (50%) and IV(62%)) as compared to sham operated animals. Chronic treatment with rofecoxib (10 and 20 mg/kg, p.o.) and nimesulide (10 and 20 mg/kg, p.o.) significantly attenuated quinolinic acid-induced behavioral and biochemical alterations as compared to quinolinic acid 300 nmol treated group. Further, rofecoxib (10, 20 mg/kg) and nimesulide (20 mg/kg) significantly restored mitochondrial enzyme complex activities in striatum as compared to quinolinic acid 300 nmol treated group. Present study highlights the therapeutic potential of cyclooxygenase inhibitors against quinolinic acid induced neurotoxicity.

Effect of complexes of precursors and modulators of coenzyme q biosynthesis on functional state of old rats' heart mitochondria

Our research demonstrate that ageing leads to changes in activity of electron-transporting enzyme complexes in myocardial mitochondria of old rats and to increased sensitivity of mitochondrial permeability transition pore to inductors of its opening - Ca2+ and phenylarsine oxide. We also observed activation of lipid and protein free-radical peroxidation processes. Administration of a complex of biologically active substances that included precursors and modulators of coenzyme Q biosynthesis (α-tocopherol acetate, 4-hydroxybenzoic acid, and methionine) we observed the increase in coenzyme Q content, correction of functional activity of mitochondrial electron-transport chain enzyme complexes, the decrease in intensivity of lipid and protein free-radical peroxidation in the heart and the decrease in sensitivity of mitochondrial permeability transition pore to inductors of its opening. This complex may be used to treat mitochondrial dysfunction under numerous pathologies of cardiovascular system, as well as in ageing.

Naringin alleviates cognitive impairment, mitochondrial dysfunction and oxidative stress induced by d-galactose in mice

Role of mitochondrial dysfunction and oxidative stress has been well documented in aging and related disorders such as Alzheimer’s disease. Bioflavonoids have been reported to have a therapeutic potential against several age related processes. Bioflavonoids are being used as a neuroprotectants in the treatment of various neurological disorders including aging. Therefore, present study has been conducted in order to explore the possible role of naringin against d-galactose induced cognitive dysfunction, oxidative damage and mitochondrial dysfunction in mice. Chronic administration of d-galactose (100 mg/kg) for 6 weeks significantly impaired cognitive performance (both in Morris water maze and elevated plus maze), locomotor activity, oxidative defense and mitochondrial complex (I, II and III) enzymes activities as compared to sham group. Six weeks naringin (40 and 80 mg/kg) treatment significantly improved cognitive performance, oxidative defense and restored mitochondria complex enzyme activities as compared to control ( d-galactose). Naringin treatment significantly attenuated acetylcholine esterase activity in d-galactose treated mice. In conclusion, present study highlights the potential role of naringin against d-galactose induced cognitive impairment, biochemical and mitochondrial dysfunction in mice.

The effect of complexes of precursors and modulators of coenzyme Q biosynthesis on the functional state of heart mitochondria of old rats

Aging is accompanied by changes in activity of electron-transport enzyme complexes in myocardial mitochondria of old rats and by increased sensitivity of the mitochondrial permeability transition pore (MPTP) to inductors of its opening (Ca2+ and phenylarsine oxide). We also observed activation of lipid and protein free-radical peroxidation processes. Administration of a complex of biologically active substances that included precursors and modulators of coenzyme Q biosynthesis (α-tocopherol acetate, 4-hydroxybenzoic acid, and methionine) caused the increase in coenzyme Q content, correction of functional activity of mitochondrial electron-transport chain enzyme complexes, the decrease in intensity of lipid and protein free-radical peroxidation in the heart mitochondria and the decrease in sensitivity of mitochondrial permeability transition pore to inductors of its opening. This complex may be recommended for treatment of mitochondrial dysfunction in various pathologies of cardiovascular system, including in aging.

Protective effect of hesperidin and naringin against 3-nitropropionic acid induced Huntington's like symptoms in rats: Possible role of nitric oxide

3-Nitropropionic acid (3-NP) is a well known experimental model to study Huntington's disease (HD) and associated neuropsychiatric problems. Present study has been designed to explore the protective effects of hesperidin, naringin, and their nitric oxide mechanism (if any) against 3-nitropropionic acid induced neurotoxicity in rats. Systemic 3-nitropropionic acid (10 mg/kg) treatment for 14 days in rats significantly induced HD like symptoms in rats as indicated by reduced locomotor activity, body weight, grip strength, oxidative defense and mitochondrial complex enzymes (complex-I, -II, and -IV) activities in striatum. Naringin and hesperidin pretreatment significantly attenuated behavioral alterations, oxidative stress and mitochondrial enzymes complex dysfunction in 3-NP treated group. l-Arginine (50 mg/kg) pretreatment with lower dose of hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly attenuated the protective effect of hesperidin and naringin respectively. Whereas l-NAME (10 mg/kg), a non-selective NOS inhibitor pretreatment with hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly potentiated their protective effect which was significant as compared to their effect per se. Study highlights the therapeutic potential of hesperidin and naringin against Huntington's like conditions and further indicates that these drugs might act through nitric oxide mechanism.

Effect of caffeic acid and rofecoxib and their combination against intrastriatal quinolinic acid induced oxidative damage, mitochondrial and histological alterations in rats

Oxidative stress has long been implicated in the neurotoxic effects of glutamate acting through N-methyl-D-aspartate (NMDA) receptors. Therefore, present study has been designed to explore the effect of rofecoxib and caffeic acid on the involvement of oxidative stress, mitochondrial dysfunction and neuronal linked with NMDA receptor-mediated excitotoxicity. Caffeic acid, is a well-known antioxidant flavanoid, implicate anti-inflammatory and immunomodulatory like actions. The present study is an attempt to investigate the antioxidant-like effect of caffeic acid and rofecoxib and their combination against QA-induced oxidative damage, mitochondrial dysfunction and histological alterations. Intrastriatal injection of quinolinic acid (300 nmol) significantly increased oxidative stress (raised lipid peroxidation, nitrite concentration, depleted SOD and catalase), altered mitochondrial complex enzyme activities and histological alteration in the ex vivo striatum. Caffeic acid (5 and 10 mg/kg, p.o.) and rofecoxib (10 and 20 mg/kg, p.o.) treatment for 21 days significantly attenuated oxidative damage and impairment in mitochondrial activities of complex enzymes in the ex vivo striatum. Further, combination of sub effective doses of rofecoxib (10 mg/kg, p.o.) and caffeic acid (5 mg/kg, p.o.) potentiated their protective effect which was significant as compared to their effect per se. The present study suggests the therapeutic effect of caffeic acid and rofecoxib combination against QA-induced ex vivo oxidative damage, mitochondrial and histological alterations in rats.

Role‐reversal of gender‐associated mitochondrial DNA affects mitochondrial function in Mytilus edulis (Bivalvia: Mytilidae)

Mussel species of the genus Mytilus possess an unusual system of mitochondrial DNA (mtDNA) transmission termed doubly uniparental inheritance. They are characterized by the presence of two highly divergent gender-associated mtDNA genomes (often with>20 and>10% divergences in DNA and amino acid sequences, respectively) that are inherited either maternally (F mtDNA) or paternally (M mtDNA). Females are typically homoplasmic for the F mtDNA and males are heteroplasmic with the F mtDNA being most common in all tissues except the gonad that is dominated by the M mtDNA. Collectively, males are polymorphic for two classes of M mtDNAs known as the "standard male" and "recently masculinized" M types (SM and RM, respectively). The coding portions of the RM mtDNA genome differ from the SM mtDNA by as much as the maternally inherited F mtDNA genome differs from the SM type. Because the SM and RM types exhibit considerable amino acid sequence divergence, we hypothesized that these differences could affect mitochondrial respiratory chain complex enzyme activities. To test this hypothesis, the activity of the major mitochondrial respiratory chain complexes (complexes II, I+III and IV) as well as the activity of citrate synthase were measured on gonad samples from males containing either the SM or RM mtDNA. Our data demonstrate that the mitochondrial subunits encoded by the RM mtDNA are associated with higher enzymatic activities than the gene products of the SM mtDNA.

Sensory ataxic neuropathy with ophthalmoparesis caused by POLG mutations

Mutations in POLG gene are responsible for a wide spectrum of clinical disorders with altered mitochondrial DNA (mtDNA) integrity, including mtDNA multiple deletions and depletion. Sensory ataxic neuropathy with ophthalmoparesis (SANDO) caused by mutations in POLG gene, fulfilling the clinical triad of sensory ataxic neuropathy, dysarthria and/or dysphagia and ophthalmoparesis, has described in a few reports. Here we described five cases of adult onset autosomal recessive sensory ataxic neuropathy with ophthalmoplegia. All patients had ataxia, neuropathy, myopathy, and progressive external ophthalmoplegia (PEO). The muscle pathology revealed ragged-red and cytochrome c oxidase (COX) negative fibers in three patients. However, deficiencies in the activities of mitochondrial respiratory chain enzyme complexes were not detected in any of the patients’ muscle samples. Multiple deletions of mtDNA were detected in blood and muscle specimens but mtDNA depletion was not found. Due to these diagnostic difficulties, POLG-related syndromes are definitively diagnosed based on the presence of deleterious mutations in the POLG gene.

Autophagocytosis of Mitochondria Is Prominent in Alzheimer Disease

Mitochondrial abnormalities are prominent in Alzheimer disease. In this study, 2 mitochondrial markers, cytochrome oxidase-1 and lipoic acid, a sulfur-containing cofactor required for the activity of several mitochondrial enzyme complexes, were compared using light and electron microscopic analyses and immunoblot assays. Both lipoic acid and cytochrome oxidase-1 immunoreactivity are increased in the cytoplasm of pyramidal neurons in Alzheimer disease compared with control cases. Of significance, lipoic acid was found to be strongly associated with granular structures, and ultrastructure analysis showed localization to mitochondria, cytosol, and, importantly, in organelles identified as autophagic vacuoles and lipofuscin in Alzheimer disease but not control cases. Cytochrome oxidase-1 immunoreactivity was limited to mitochondria and cytosol in both Alzheimer and control cases. These data suggest that mitochondria are key targets of increased autophagic degradation in Alzheimer disease. Whether increased autophagocytosis is a consequence of an increased turnover of mitochondria or whether the mitochondria in Alzheimer disease are more susceptible to autophagy remains to be resolved.

Mitochondrial Respiratory Complex I Deficiency Simulating Spinal Muscular Atrophy

Two female patients with clinical features resembling spinal muscular atrophy were presented. Patient 1 presented with hypotonia and proximal weakness of extremities at age 4 months. Electromyography revealed motor neuronopathy suggestive of spinal muscular atrophy. Patient 2 presented with severe hypotonia, motor weakness, and joint contractures since birth. Muscle biopsy findings were consistent with spinal muscular atrophy. However, deletions in the survival motor neuron gene and the neuronal apoptosis inhibitor protein gene were not found in both patients. They finally manifested clinical features unlike spinal muscular atrophy: epileptic seizure, cardiomyopathy, and spasticity. The clinical course of each patient was not like that of spinal muscular atrophy type I. Mitochondrial respiratory chain complex enzyme activities in cultured skin fibroblasts were measured. Respiratory complex I enzyme activity was decreased, suggestive of isolated complex I deficiency in both patients. In conclusion, in patients who have clinical features resembling spinal muscular atrophy but no deletions in the spinal muscular atrophy gene, the possibility of the mitochondrial respiratory chain complex I deficiency should be considered.

Impaired Activity of Mitochondrial Respiratory Chain Enzyme Complexes and Mitochondrial Genomic Aberrations in Leukemia Cells from Patients with Acute Myeloid Leukemia.

Background: Mitochondrial DNA (mtDNA) was particularly susceptible to oxidative damage and mutation because of the high rate of reactive oxygen species (ROS) generation and inefficient mtDNA repair system. Somatic mutations of mtDNA lead to errors in the mtDNA-encoded polypeptide chains belonging to the proton-translocating complexes of the mitochondrial inner membrane. Therefore, we investigated level of ROS, mitochondrial genomic aberrations (mitochondrial genomic instability, mtGI) and the change in enzyme activity of mitochondrial respiratory chain complexes I–IV and in acute myeloid leukemia (AML) cells.Methods: Forty-eight AML bone marrow samples and 57 control blood samples were used after receiving informed consent. The intracellular ROS generation of cells can be investigated using the 2′,7′-dichlorfl uorescein-diacetate and flow cytometry. The results were expressed as mean fluorescence intensity (MFI). We sequenced the mtDNA control region, the tRNA leucine 1 gene plus part of NADH dehydrogenase (ND) 1, and cytochrome b (CYTB) directly and checked the activity of mitochondrial respiratory chain enzyme complexes I to IV using a spectrophotometric kinetic assay. To investigate mtGI, we also examined six mtGI repeats (303 poly C, 16184 poly C, 514CA repeats, 3566 poly C, 12385 poly C and 12418 poly A) using size-based PCR product separation with capillary electrophoresis. mtGI was further confirmed by cloning and sequencing. The mtDNA molecules were analyzed quantitatively using real-time PCR.Results: MFI in AML cells (4,435±709) was significantly higher than those in control blood cells (1,562±141) (P<0.05). Many polymorphisms, as well as new mtDNA variants in the control region and ND1 and CYTB genes were detected in this study. A total of 606 mtDNA sequence variants were identified. Of these, 15 mtDNA variants were identified as novel mutations that were absent from control subjects and established mtDNA polymorphism databases. There were profound alterations of mtGI in the 303 poly C, 16184 poly C, and 514 CA repeats. Seven patients (15%) had leukemia cell-specific mtDNA substitution mutations in the ND1 and CYTB genes. The enzyme activities of AML cells compared with control subjects were 0.828±2.72 vs. −2.48±3.18 for complex I, 0.03±0.08 vs. 0.144±1.72 for complex II/III, and 0.244±0.16 vs. 0.505±0.73 for complex IV. The AML cells showed decreased enzyme activity in respiratory chain complexes I, II, and III. The AML cells had an approximately two-fold decrease in mtDNA copy number compared with the results for control subjects.Conclusion: Mitochondrial genomic aberrations were commonly observed in primary AML cells. High level of ROS in these cells might be an injury to the mitochondrial genome. These mtDNA alterations may impair the activity of mitochondrial respiratory chain enzyme complexes and reduce the mtDNA copy number in patients with AML.

Aerobic training is safe and improves exercise capacity in patients with mitochondrial myopathy

Exercise intolerance is a prominent symptom in patients with mitochondrial myopathy (MM), but it is still unsettled whether exercise training is safe and beneficial for patients with MM. To address this, we studied the effect of 12 weeks cycle training on exercise capacity, quality of life and underlying molecular and cellular events in five patients with single large-scale deletions, one with a microdeletion and 14 with point mutations of mitochondrial DNA (mtDNA), and 13 healthy subjects. Each training session lasted 30 min, and was performed at an intensity of 70% of VO2max (maximal oxygen uptake). Each subject performed 50 training sessions in 12 weeks. All subjects were evaluated before and after training, and 13 MM patients were studied after 8 weeks of deconditioning. Evaluation included VO2max and mutation load and mtDNA quantity, mitochondrial enzymatic activity, and number of centrally nucleated, apoptotic, ragged red and cytochrome oxidase (COX)-negative fibres in muscle biopsies from the quadriceps muscle. After 12 weeks of training, VO2max and muscle citrate synthase increased in MM (26 and 67%) and healthy (17 and 65%) subjects, while mtDNA quantity in muscle only increased in the MM patients (81%). In the MM patients, training did not change mtDNA mutation load in muscle, mitochondrial enzyme complex activities, muscle morphology and plasma creatine kinase. After deconditioning, VO2max and citrate synthase activity returned to values before training, while muscle mtDNA mutation load decreased. These findings show that aerobic training efficiently improves oxidative capacity in MM patients. Based on unchanged levels of mutant load in muscle, morphological findings on muscle biopsy and plasma creatine kinase levels during training, the treatment appears to be safe. Regular, supervised aerobic exercise is therefore recommended in MM patients with the studied mutations.

Overexpression of Mitochondrial Peroxiredoxin-3 Prevents Left Ventricular Remodeling and Failure After Myocardial Infarction in Mice

Mitochondrial oxidative stress and damage play major roles in the development and progression of left ventricular (LV) remodeling and failure after myocardial infarction (MI). We hypothesized that overexpression of the mitochondrial antioxidant, peroxiredoxin-3 (Prx-3), could attenuate this deleterious process. We created MI in 12- to 16-week-old, male Prx-3-transgenic mice (TG+MI, n=37) and nontransgenic wild-type mice (WT+MI, n=39) by ligating the left coronary artery. Prx-3 protein levels were 1.8 times higher in the hearts from TG than WT mice, with no significant changes in other antioxidant enzymes. At 4 weeks after MI, LV thiobarbituric acid-reactive substances in the mitochondria were significantly lower in TG+MI than in WT+MI mice (mean+/-SEM, 1.5+/-0.2 vs 2.2+/-0.2 nmol/mg protein; n=8 each, P<0.05). LV cavity dilatation and dysfunction were attenuated in TG+MI compared with WT+MI mice, with no significant differences in infarct size (56+/-1% vs 55+/-1%; n=6 each, P=NS) and aortic pressure between groups. Mean LV end-diastolic pressures and lung weights in TG+MI mice were also larger than those in WT+sham-operated mice but smaller than those in WT+MI mice. Improvement in LV function in TG+MI mice was accompanied by a decrease in myocyte hypertrophy, interstitial fibrosis, and apoptosis in the noninfarcted LV. Mitochondrial DNA copy number and complex enzyme activities were significantly decreased in WT+MI mice, and this decrease was also ameliorated in TG+MI mice. Overexpression of Prx-3 inhibited LV remodeling and failure after MI. Therapies designed to interfere with mitochondrial oxidative stress including the antioxidant Prx-3 might be beneficial in preventing cardiac failure.

Automated Spectrophotometric Analysis of Mitochondrial Respiratory Chain Complex Enzyme Activities in Cultured Skin Fibroblasts

Mitochondrial respiratory chain complex (RCC) disorders may occur as commonly as 1 in 8500 individuals. Because of the great variability of phenotypic presentations, measurement of individual RCC enzyme activities is a crucial diagnostic process. Current assay methods are time-consuming and labor-intensive and thus constitute a major impediment to clinical practice. A method with a faster turnaround time would therefore be beneficial. We developed an automated spectrophotometric method for measuring the respiratory chain enzyme activities of complex I, complex II + III, and complex IV with the Hitachi 912, an automated spectrophotometer. Mitochondrial citrate synthase was also determined for normalization of the RCC activities. A blinded method comparison with samples from an external testing center yielded a 91% concordance of interpretations. Mean intraassay imprecision (as CV; n = 20) in a single batch analysis of each RCC was 5.9%. Interassay imprecision, evaluated on 2 samples harvested and analyzed 3 times each, gave mean CVs of 10%-18%. With this automated method, a panel of RCC enzyme activities can be determined in <2 h. In addition, an immunoblot assay using monoclonal antibodies against specific subunits of RCC enzyme complexes can be informative in cases of borderline enzyme activity. Our results suggest that in vitro diagnosis of RCC enzyme deficiencies in skin fibroblasts is an effective alternative to invasive muscle biopsy.

Overexpression of Mitochondrial Transcription Factor A Ameliorates Mitochondrial Deficiencies and Cardiac Failure After Myocardial Infarction

Mitochondrial DNA (mtDNA) copy number is decreased not only in mtDNA-mutation diseases but also in a wide variety of acquired degenerative and ischemic diseases. Mitochondrial transcription factor A (TFAM) is essential for mtDNA transcription and replication. Myocardial mtDNA copy number and TFAM expression both decreased in cardiac failure. However, the functional significance of TFAM has not been established in this disease state. We have now addressed this question by creating transgenic (Tg) mice that overexpress human TFAM gene and examined whether TFAM could protect the heart from mtDNA deficiencies and attenuate left ventricular (LV) remodeling and failure after myocardial infarction (MI) created by ligating the left coronary artery. TFAM overexpression could ameliorate the decrease in mtDNA copy number and mitochondrial complex enzyme activities in post-MI hearts. Survival rate during 4 weeks of MI was significantly higher in Tg-MI than in wild-type (WT) littermates (WT-MI), although infarct size was comparable. LV cavity dilatation and dysfunction were significantly attenuated in Tg-MI. LV end-diastolic pressure was increased in WT-MI, and it was also reduced in Tg-MI. Improvement of LV function in Tg-MI was accompanied by a decrease in myocyte hypertrophy, apoptosis, and interstitial fibrosis as well as oxidative stress in the noninfarcted LV. Overexpression of TFAM inhibited LV remodeling after MI. TFAM may provide a novel therapeutic strategy of cardiac failure.

Polarographic evaluation of mitochondrial enzymes activity in isolated mitochondria and in permeabilized human muscle cells with inherited mitochondrial defects.

Inherited disturbances of the mitochondrial energy generating system represent a heterogeneous group of disorders associated with a broad spectrum of metabolic abnormalities and clinical symptoms. We used the polarographic and spectrophotometric method for detection of mitochondrial disorders, because these two techniques provide a different insight into mitochondrial function. In six patients suspected of mitochondrial disease we found defects of complex I (two patients), complex III (one patient), complex IV (two patients) and a combination of defect of complex III and IV (one patient). Citrate synthase activity, used as the reference enzyme, was not changed. A comparison of the two methods showed several differences in evaluation of mitochondrial enzymes activity due to the fact that both methods used different conditions for enzyme activity measurements. In contrast to oxygen consumption measurements, where the function of the whole-integrated respiratory chain is characterized, spectrophotometric measurements characterize activities of isolated complexes in disintegrated membranes. However, it may be concluded from our experiments that both methods provide useful and complementary data about mitochondrial energetic functions. Whereas spectrophotometric data are suitable for evaluation of maximal enzyme activities of mitochondrial enzyme complexes, polarographic data provide better information about enzyme activities in cells with mitochondrial defects under in situ conditions.

A cholesterol-binding and transporting protein from rat liver mitochondria

In this communication, we present results indicating a protein isolated from rat liver mitochondrial intermembrane space that is capable of binding cholesterol and transporting it between the inner and outer mitochondrial membranes. This protein has a molecular weight of 57.5 kDa by SDS-PAGE; however, under native conditions, there is cholesterol-binding capability only as a 115 kDa dimer. Our data show that this dimeric protein may play a role in the regulation of mitochondrial membrane cholesterol levels, a perquisite for the optimal activity of inner mitochondrial membrane-associated enzyme complexes. In addition, it appears that this protein is largely responsible for the differences in membrane cholesterol levels observed in normal and hepatoma mitochondria, a discrepancy which may help to explain the lack of energy production via oxidative phosporylation in malignant tumor mitochondria.

Oxidative damage to mitochondrial DNA and activity of mitochondrial enzymes in chronic active lesions of multiple sclerosis

Soluble products of activated immune cells include reactive oxygen species (ROS) and nitric oxide (NO) with a high potential to induce biochemical modifications and degenerative changes in areas of inflammation in the central nervous system (CNS). Previously, we demonstrated an increased production of ROS by activated mononuclear cells (MNC) of patients with multiple sclerosis (MS) compared to those of controls, and development of oxidative damage to total DNA in association with inflammation in chronic active plaques. The current study aimed to determine whether mitochondrial (mt)DNA is affected by oxidative damage, and whether oxidative damage to mitochondrial macromolecules (including mtDNA) is associated with a decline in the activity of mitochondrial enzyme complexes. Using molecular and biochemical methods we demonstrate a trend for impaired NADH dehydrogenase (DH) activity and a possible compensatory increase in complex IV activity in association with oxidative damage to mtDNA in chronic active plaques. Immunohistochemistry confirms the increase of oxidative damage to DNA predominantly located in the cytoplasmic compartment of cells in chronic active plaques. These observations suggest that oxidative damage to macromolecules develops in association with inflammation in the CNS, and may contribute to a decline of energy metabolism in affected cells. As observed in neurodegenerative diseases of non-inflammatory origin, decreased ATP synthesis can ultimately lead to cell death or degeneration. Therefore, elucidation of this pathway in MS deserves further studies which may identify neuroprotective strategies to prevent tissue degeneration and the associated clinical disability.

A selective defect of cytochrome c oxidase is present in brain of Alzheimer disease patients

To assess mitochondrial function and test the hypothesis of an underlying oxidative phosphorylation defect in Alzheimer disease (AD), we evaluated the activities of mitochondrial respiratory chain enzyme complexes I+III, complexes II+III, complex IV (cytochrome c oxidase, COX), succinate dehydrogenase, and citrate synthase in the frontal cortex, temporal cortex, hippocampus, and cerebellum of 23 AD patients and 13 normal human brains. The major finding was a significant decrease in COX activity in AD temporal cortex and hippocampus, both whether activities were expressed per noncollagen protein content (49 ± 4.6 versus 78 ± 10.8 nmol/min/mg NCP, P = 0.006; 23 ± 1.9 versus 48.6 ± 8.1 nmol/min/mg NCP, p = 0.003) or corrected for citrate synthase activity (1.6 ± 0.2 versus 3 ± 0.4, P = 0.001; 0.76 ± 0.1 versus 1.76 ± 0.26, P = 0.0009). There were no significant differences in the activities of complexes I+III, II+III, and of succinate dehydrogenase in any of the brain regions examined. Our results suggest a specific defect of COX in the AD brain versus the normal human brain, which may contribute to impaired energy generation. Biochemically, the defect is confined to selected brain regions, suggesting anatomic specificity.