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PubMed1995

Magnesium cardioplegia enhances mRNA levels and the maximal velocity of cytochrome oxidase I in the senescent myocardium during global ischemia.

BACKGROUND: The aged myocardium accumulates significantly more cytosolic calcium [Ca2+]i during ischemia, and functional recovery is more severely compromised as compared with the mature heart. Cardioplegia ameliorates these phenomena. The mechanism by which increased calcium accumulation reduces functional recovery in the senescent myocardium is unknown, but it has been suggested that futile calcium cycling in the mitochondria leading to depletion of ATP stores during normothermic global ischemia may be involved. METHODS AND RESULTS: To investigate the effect of cardioplegia on mitochondrial calcium ([Ca2+]mt) accumulation and the expression of cytochrome oxidase I (COX I) during global ischemia, mitochondria were isolated from mature (age, 15 to 20 weeks) and aged (age > 130 weeks) rabbit hearts after Langendorff perfusion. Five perfused heart groups were investigated: 30 minutes of global ischemia without treatment (control), with potassium (K, 20 mmol/L), magnesium (Mg, 20 mmol/L), or potassium and magnesium (K/Mg) cardioplegia. No significant difference in [Ca2+]mt was evident in mature hearts with any protocol. In aged hearts, [Ca2+]mt was increased in global ischemia but was ameliorated with Mg and K/Mg cardioplegia. COX I mRNA levels in aged hearts were lower in both control and global ischemia but were increased with cardioplegia. Maximal velocities for COX I were significantly increased with Mg cardioplegia both in the mature and the aged myocardium. CONCLUSIONS: K and/or Mg cardioplegia ameliorates [Ca2+]mt accumulation in aged hearts during normothermic global ischemia and increases COX I mRNA levels to a level not significantly different from that found in mature hearts.

Aging↗
PubMed1979

Exchangeability and rate of flip-flop of phosphatidylcholine in large unilamellar vesicles, cholate dialysis vesicles, and cytochrome oxidase vesicles.

Three model membrane systems have been characterized in terms of their interaction with phospholipid exchange proteins. Large unilamellar vesicles of phosphatidylcholine prepared by ether vaporization are shown to be homogeneous by gel filtration. Phospholipid exchange proteins from three sources are capable of catalyzing the rapid exchange of approximately half of the phospholipid from these vesicles. The remaining pool of radioactive phospholipid is virtually nonexchangeable (t1/2 of several days). Small unilamellar vesicles of phosphatidylcholine prepared by cholate dialysis also exhibit two pools of phospholipid (65% rapidly exchangable, 35% very slowly exchangeable) when incubated with beef liver phospholipid exchange protein. Cytochrome oxidase vesicles prepared both by a cholate dialysis method and by a direct incorporation method have been fractionated on a Ficoll discontinuous gradient, and tested for interaction with beef heart exchange protein. Two pools of phospholipid are once again observed (70% rapidly exchangable, 30% nonexchangeable), even for vesicles which have incorporated the transmembranous enzyme at a phospholipid to protein weight ratio of 2. The size of the rapidly exchangeable pool of phosphatidylcholine for each of the vesicle systems is consistent with the calculated fraction of phospholipid in the outer monolayer. The extremely slow rate of exchange of the second pool of the second pool of phospholipid reflects the virtual nonexistence of phospholipid flip-flop in any of these model membranes.

Biological Transport↗
PubMed2004

Origin of intra-individual variation in PCR-amplified mitochondrial cytochrome oxidase I of Thrips tabaci (Thysanoptera: Thripidae): mitochondrial heteroplasmy or nuclear integration?

The mitochondrial genome is increasingly being used as a species diagnostic marker in insects. Typically, genomic DNA is PCR amplified and then analysed by restriction analyses or sequencing. This analysis system may cause some serious problems for molecular diagnosis. Besides the errors introduced by the PCR process, mtDNA sequence variation of amplified fragments may originate from mtDNA heteroplasmy or from nuclear integrations of mtDNA fragments, both of which have been shown to occur in insects. Here we document abundant variation in PCR-amplified sequences of the mitochondrial cytochrome oxidase I gene of Thrips tabaci. We confirm that the most common haplotype is of mitochondrial origin. Some of the observed mutations were introduced by the amplification process. However, the occurrence of some haplotypes at elevated frequencies indicates that within-individual variation of the respective fragment exists at low levels in T. tabaci. The frequencies of these sequences are too low to negatively affect mtDNA-based molecular diagnosis of T. tabaci. The possible origin of these variant haplotypes is discussed.

Animals↗
PubMed2003

Mitochondrial DNA cytochrome oxidase I gene: potential for distinction between immature stages of some forensically important fly species (Diptera) in western Australia.

Forensic entomology requires the fast and accurate identification of insects collected from a corpse for estimation of the postmortem interval (PMI). Identification of specimens is traditionally performed using morphological features of the insect. Morphological identification may be complicated however by the numerical diversity of species and physical similarity between different species, particularly in immature stages. In this study, sequencing was performed to study the mitochondrial DNA (mtDNA) as the prospective basis of a diagnostic technique. The sequencing focused on a section of the cytochrome oxidase I encoding region of mtDNA. Three species of calliphorid (blow flies) commonly associated with corpses in western Australia, Calliphora dubia, Chrysomya rufifacies and Lucilia sericata, in addition to specimens of Calliphora augur and Chrysomya megacephala were studied. Phylogenetic analysis of data revealed grouping of species according to genus. The DNA region sequenced allowed identification of all species, providing high support for separation on congeneric species. Low levels of variation between some species of the same genus however indicate that further sequencing is required to locate a region for development of a molecular-based technique for identification.

Animals↗
PubMed2004

Recent African derivation of Chrysomya putoria from C. chloropyga and mitochondrial DNA paraphyly of cytochrome oxidase subunit one in blowflies of forensic importance.

Chrysomya chloropyga (Wiedemann) and C. putoria (Wiedemann) (Diptera: Calliphoridae) are closely related Afrotropical blowflies that breed in carrion and latrines, reaching high density in association with humans and spreading to other continents. In some cases of human death, Chyrsomya specimens provide forensic clues. Because the immature stages of such flies are often difficult to identify taxonomically, it is useful to develop DNA-based tests for specimen identification. Therefore we attempted to distinguish between C. chloropyga and C. putoria using mitochondrial DNA (mtDNA) sequence data from a 593-bp region of the gene for cytochrome oxidase subunit one (COI). Twelve specimens from each species yielded a total of five haplotypes, none being unique to C. putoria. Therefore it was not possible to distinguish between the two species using this locus. Maximum parsimony analysis indicated paraphyletic C. chloropyga mtDNA with C. putoria nested therein. Based on these and previously published data, we infer that C. putoria diverged very recently from C. chloropyga.

Animals↗
PubMed2002

Metabolic changes in the basal ganglia of patients with Huntington's disease: an in situ hybridization study of cytochrome oxidase subunit I mRNA.

On the basis of the functional model of the basal ganglia developed in the 1980s and the neuropathological findings in Huntington's disease (HD), changes in the neuronal activity of the basal ganglia have previously been proposed to explain the abnormal movements observed in this pathology. In particular, it has been stated that the neurodegenerative process affecting the basal ganglia in the disease should provoke a hypoactivity in the internal segment of the pallidum (GPi) that could explain choreic movements observed in the disease. To test this functional hypothesis, we performed an in situ hybridization study on control and HD brains postmortem, taking cytochrome oxidase subunit I (COI) mRNAs expression as index of neuronal activity. As most of the HD patients studied were under chronic neuroleptic (NL) treatment, we also studied the brains of non-HD patients under chronic NL treatment. Our results show that in HD brain the number of neurons expressing COI mRNA tends to be lower in the striatum, GPe and GPi, suggesting a severe involvement of these structures during the neurodegenerative process. Moreover, COI mRNA level of expression was markedly reduced within neurons of the putamen and GPe. Surprisingly, COI mRNA expression was not modified in the GPi in HD brains compared with controls. This paradoxical result in the GPi may be explained by the antagonistic effect of GPe hypoactivity and the degenerative process involving neurons of GPi. Our results indicate that the functional modifications, and consequently the pathophysiology of abnormal movements, observed in HD basal ganglia are more complex than expected from the currently accepted model of the basal ganglia organization.

Adult↗
PubMed1977

[Effect of thyrotropin releasing hormone thyroxine on the activity of cytochrome oxidase in rat adenohypophysis].

Thyrotropin releasing-hormone (TRH) increased the activity of cytrochrome C oxidase in a concentration of 0.01 and 1.0 microgram/ml in the adenohypophysis of rats fed methylthiouracil for 6 weeks. This effect of TRH on the activity of the enzyme was blocked with T4 added to the incubation medium in a concentration of 20 microgram/ml. Actinomycin D (20 MICrogram/ml) prevented the block of the enzyme with thyroxin. In a concentration of 0.01 microgram/ml TRH, and in a concentration of 2.0 microgram/ml T4 failed to change the activity of cytochrome oxidase in the adenohypophysis of normal and partially thyroidectomized rats.

Animals↗
PubMed2005

Molecular characterization of closely related species in the parasitic genus Encarsia (Hymenoptera: Aphelinidae) based on the mitochondrial cytochrome oxidase subunit I gene.

The genus Encarsia Förster includes parasitoid species that are effective natural enemies of whitefly and armoured scale insect agricultural pests. Within this genus, several species groups have been recognized on the basis of morphological similarity, although their monophyly appears uncertain. It is often difficult to separate morphologically similar species, and there is evidence that some species could in fact be complexes of cryptic species. Their correct identification is fundamental for biological control purposes. Recently, due to unreliability of morphological characters, molecular techniques have been investigated to identify markers that differentiate closely related species. In this study, DNA variation in an approximately 900 bp segment of the mitochondrial cytochrome oxidase subunit I (COI) gene was examined by both sequencing and PCR-RFLP. Two pairs of species that are difficult to distinguish morphologically were analysed: Encarsia formosa Gahan and Encarsialuteola Howard, belonging to the luteola group, and two populations of Encarsiasophia (Girault & Dodd) from Pakistan and Spain, belonging to the strenua group, recently characterized as cryptic species. High sequence divergence and species-specific restriction patterns clearly differentiate both species pairs. Parsimony analysis of the nucleotide sequences was also performed, including Encarsiahispida De Santis (luteola group) and Encarsia protransvena Viggiani (strenua group). Two monophyletic clades supporting the two groups of species considered were resolved. The results of this study support the use of the COI gene as a useful marker in separating species of Encarsia, for which morphological differences are subtle. Moreover, the COI gene appears potentially useful for understanding phylogenetic relationships in this genus.

Animals↗
PubMed2005

Differentiation of Culicoides obsoletus and Culicoides scoticus (Diptera: Ceratopogonidae) based on mitochondrial cytochrome oxidase subunit I.

Culicoides obsoletus (Meigen) and Culicoides scoticus Downes & Kettle (Diptera: Ceratopogonidae) are sibling species of the Obsoletus group. This group comprises species of biting midges that are suspect vectors of bluetongue virus (family Reoviridae, genus Orbivirus, BTV) and African horse sickness virus (family Reoviridae, genus Orbivirus, AHSV). BTV and AHSV have been isolated several times from females of this group, although it has not been possible to determine the particular species harboring the virus, because of the inability to clearly identify the females of each species based on morphology. Both sexes of C. obsoletus and C. scoticus midges from Catalonia and the Balearics (Spain) were sequenced for the mitochondrial gene cytochrome oxidase subunit I (COI), and these sequences were analyzed to determine intra- and interspecific genetic variability. Species-specific primers for C. obsoletus and C. scoticus were designed and a polymerase chain reaction (PCR)-based diagnostic assay based on the COI gene and using a hemi-nested PCR technique was developed for reliably distinguishing the females of both species. The species-specific PCR diagnostic was compared with morphological discrimination of C. obsoletus and C. scoticus females. The morphologic characters were not fully reliable.

African Horse Sickness↗
PubMed1993

Time-resolved study of tryptophan fluorescence in vesicle reconstituted cytochrome oxidase. Effect of redox transition.

Time-resolved study of fluorescence decay of the tryptophan residue in bovine cytochrome c oxidase in phospholipid vesicles is reported for the first time. The effect of the redox state of the protein on its conformation has been investigated using time-resolved decay of tryptophan fluorescence in the oxidised and reduced protein. The fluorescence decay was best fitted using a discrete three exponential model. Amplitude distribution of lifetimes also showed three distinct regions in the analysis of decay profiles by the maximum entropy method (MEM). Results of the time resolved studies showed that the amplitudes as well as the lifetimes of the tryptophan fluorescence remain the same for the oxidised and the reduced states of cytochrome c oxidase, indicating that the environment around tryptophan residues remains more or less unaltered on reduction of the protein. The results suggest that there is no global conformational change in the protein on electron transfer and support the possibility of the existence of local fluctuations in the protein during the redox cycle.

Animals↗
PubMed2004

Involvement of the mammillary bodies in spatial working memory revealed by cytochrome oxidase activity.

In view of the inconclusive findings relating the nuclei of the mammillary bodies (MB) with spatial memory, we evaluated the oxidative metabolic activity of the medial and lateral nuclei of the mammillary bodies (MB) after training young rats (30 days) of both sexes in the Morris water maze. Different groups were trained in spatial working (WM) or reference memory (RM) tasks, respectively. The corresponding naïve groups swam for the same amount of time as the trained groups but without the escape platform. Control groups were added that had not been manipulated in any way. No sex-related differences were detected in the working memory task although males exhibited better reference memory than females. Cytochrome oxidase (CO) activity, an endogenous metabolic marker for neuronal activity, was measured in all the groups. CO activity increased significantly in both MB nuclei of male and female rats only in the spatial working memory group. In addition, high CO activity in the lateral nucleus of the MB was linearly correlated with lower escape latencies in both sexes after training in the working memory task. No CO activity changes were found in the basolateral amygdala (BL) in any of the experimental groups. This nucleus was used as a control brain region because of its participation in emotional behavior. The results suggest a specific role of the MB nuclei in spatial working memory in both sexes.

Animals↗
PubMedDate not supplied

Pattern of ocular dominance columns and cytochrome oxidase activity in a macaque monkey with naturally occurring anisometropic amblyopia.

Unilateral eyelid suture, a model for amblyopia induced by congenital cataract, produces shrinkage of the deprived eye's ocular dominance columns in the striate cortex. Loss of geniculocortical projections are thought to account for the poor vision in the amblyopic eye. It is uncertain whether ocular dominance columns become shrunken in other forms of amblyopia. We examined the striate cortex in a pigtailed macaque with natural anisometropia discovered at age 5 months. Amblyopia in the left eye was documented at 1 year by behavioral testing. At age 6 years, the left eye was injected with [3H]proline and the striate cortex was processed for autoradiography and cytochrome oxidase (CO). The ocular dominance columns in layer IVc labelled with [3H]proline were normal. CO staining showed a novel pattern of thin dark bands in layer IV. These bands occupied the core zones at the center of the ocular dominance columns. Their appearance resulted from relative loss of CO activity along the borders of the ocular dominance columns, regions specialized for binocular processing. These findings indicate that not all forms of amblyopia are accompanied by shrinkage of ocular dominance columns. The unusual pattern of CO staining in layer IVc reflected a subtle alteration in metabolic activity which may have resulted from impairment of binocular function in anisometropic amblyopia.

Amblyopia↗
PubMed2004

Postnatal maturation of cytochrome oxidase and lactate dehydrogenase activity and age-dependent consequences of lithium-pilocarpine status epilepticus in the rat: a regional histoenzymology study.

The lithium-pilocarpine (Li-Pilo) model of epilepsy reproduces some pathophysiological, temporal, and developmental features of human temporal lobe epilepsy. In this model, rates of cerebral glucose utilization measured by the [(14)C]2-deoxyglucose technique increased during the initial status epilepticus (SE) and decreased during the latent or chronic periods. To correlate these metabolic changes with the activities of the enzymes of the glycolytic and tricarboxylic acid cycle pathways, we measured by histoenzymology the regional activity of two key enzymes of glucose metabolism, lactate dehydrogenase (LDH) for the anaerobic pathway and cytochrome oxidase (CO) for the aerobic pathway coupled to oxidative phosphorylation, at various times after SE induced by Li-Pilo in 10- (P10), 21-d-old (P21) and adult rats for CO and in adult rats only for LDH. CO activity was slightly affected in P10 and P21 rats only at 4 and 24 h and normalized by 14 d after SE. In adult rats, CO activity decreased at 4 and 24 h in damaged areas, like entorhinal cortex, hippocampal CA3 area, amygdala, and thalamus. At 14 d after SE, CO activity was decreased only in entorhinal cortex and increased in brainstem regions involved in the remote control of seizures. In adult rats, LDH activity decreased at 24 h and 14 d after SE in sensorimotor and entorhinal cortex. These data show that the enzymatic equipment underlying the metabolism of glucose is not severely affected by Li-Pilo SE and confirm our previous observations concerning the relative metabolic hyperactivity of brain regions involved in the seizure circuit despite marked neuronal loss.

Age Factors↗
PubMed1999

Effects of electrolytic lesions of the lateral pallidum on motor coordination, spatial learning, and regional brain variations of cytochrome oxidase activity in rats.

In view of recent theories suggesting a role for basal ganglia circuits in motor control and cognition, rats with bilateral electrolytic lesions of the lateral part of the globus pallidus were compared with control rats on motor coordination tasks and spatial learning in the Morris water maze. By comparison with sham-operated controls, rats with lesions of the lateral pallidum were impaired during acquisition of the rotorod task. Deficits were observed in a wooden beam task, but not in hole-board and suspended string tests. In addition, lesioned rats were impaired during acquisition of place learning but not of visuomotor guidance in the Morris water maze. Alterations of brain metabolism, as assessed by cytochrome oxidase activity, were found in three regions of lesioned rats, the subthalamic nucleus, the superior colliculus, and the centromedial thalamus of lesioned rats, probably as a result of interrupted neocortico-basal ganglia circuitry as a secondary consequence of the primary lesion.

Animals↗
PubMed1998

A quantitative cytochrome oxidase mapping study, cross-regional and neurobehavioural correlations in the anterior forebrain of an animal model of Attention Deficit Hyperactivity Disorder.

The aim of this study was to trace by molecular imaging techniques the neural substrates of attention deficit hyperactivity disorder (ADHD) using the spontaneously hypertensive rat (SHR) as animal model. Adult SHR and Wistar-Kyoto (WKY) controls were used throughout this study. In experiment 1, naive male SHR and WKY were used, whereas in experiment 2 SHR and WKY rats of both genders were trained on a multiple fixed interval (FI (120 s for water, 5-min extinction)) paradigm and sacrificed 6 months later. In both experiments coronal sections of the anterior forebrain were processed for quantitative cytochrome oxidase (COase) histochemistry by the method of Gonzalez-Lima. Optical density values were transformed into actual enzyme activity units by using tissue-calibrated standards. In experiment 1, non-trained male rats of the SHR line showed lower COase activity in the medial and lateral prefrontal cortices, compared with WKY controls. In experiment 2, there was a line x treatment interaction effect in the pole of the nucleus accumbens (ACB). Regional correlative analyses revealed that: (i) under basal conditions, SHR are more synchronized than WKY rats in the COase level of different brain regions; and (ii) the training desynchronizes COase activity in the WKY, further synchronizes it and increases the cross-talk between hemispheres in male SHR only. Neurobehavioral covariations between behavioural scores and metabolic capacity in the medial and lateral prefrontal/frontal cortices, the caudate-putamen complex (CPU), the pole, core, and shell of the accumbal complex (ACB), and the ventral pallidum (VP), indicated that, in the WKY rats, the frequency of lever pressing covaried positively with the COase activity in the CPU, whereas in the SHR covaried with both medial and lateral prefrontal/frontal cortices. The bursts of activity during the 1-1.33-s segment was positively correlated, in the WKY rats only, with the core and shell of the ACB, and with the VP. Finally, the correlative profiles showed significant gender differences with effects in male SHR only. Thus, the results lend support to the involvement of the cortico-striato-pallidal system in ADHD.

Animals↗
PubMed1997

Phylogeny of the genus Rhagoletis (Diptera: Tephritidae) inferred from DNA sequences of mitochondrial cytochrome oxidase II.

Despite a considerable literature describing the biology of flies in the tephritid genus Rhagoletis, the phylogenetic relationships of the more than 60 species classified within the genus are not well resolved. Knowledge of these relationships is important, not only in terms of obtaining information that will be useful for the control of Rhagoletis where it is an agricultural pest, but also in determining what role host plant shifts have played in the generation of Rhagoletis species diversity, a focal point in debate over the role of sympatric speciation in the evolutionary process. In this paper, the phylogenetic history of 28 Rhagoletis species and 6 species in related tephritid genera is inferred from nucleotide sequences of subunit II of the mitochondrial cytochrome oxidase (COII) gene and the adjacent tRNALeu/COII intergenic region. Analyses of the data using distance and character-state approaches suggest the following: (i) the genus Rhagoletis as currently defined is not monophyletic; (ii) the 5 predominantly North American Rhagoletis species groups do constitute a monophyletic assemblage; (iii) the North American species groups form 2 clades, 1 consisting of taxa in the R. pomonella and R. tabellaria species groups (plus R. fausta), and the other consisting of taxa in the R. ribicola, R. cingulata, and R. suavis species groups; and (iv) the origin(s) of the North American species groups is obscure, as evidenced by the recovery of clades containing both Palearctic and Neotropical taxa. Areas of congruence and conflict with published phylogenies of Rhagoletis are examined and, while many areas of conflict may be due to an insufficient number of characters or incomplete taxon sampling, we cannot discount the possibility that real differences exist between the Rhagoletis mitochondrial DNA gene tree and the Rhagoletis species tree.

Animals↗
PubMed1991

Ultrastructural localization of cytochrome oxidase and monoamine oxidase on microcylinders, a Long-Evans rat-specific mitochondrial inclusion.

The presence of a unique inclusion body, the microcylinder, in the intracristal space of mitochondria was previously reported in various types of cells from spotted rats of the Long-Evans strain, but was not found in cells of albino rats. The microcylinder is about 30 nm in diameter and of indefinite length, and is composed of six filamentous subunits surrounding a central one. We performed electron microscopic cytochemical studies on the cells of uriniferous tubules and the corpus striatum in normal spotted rats of the Long-Evans strain and albino rats of Wistar and Sprague-Dawley strains. On the basis of oxidative polymerization of 3, 3'-diaminobenzidine by cytochrome oxidase (CYO) an cupric ferrocyanide deposition by monoamine oxidase (MAO), microcylinders were demonstrated to exhibit activity of these enzymes. Reaction products of other mitochondrial enzymes, such as succinate dehydrogenase and lactate dehydrogenase, were not deposited on microcylinders. We conclude that microcylinders are rat strain-specific mitochondrial inclusions and consist of protein components, particularly containing the mitochondrial enzymes CYO and MAO.

Animals↗
PubMed2004

The 3'-untranslated region of cytochrome oxidase II mRNA functions in RNA editing of African trypanosomes exclusively as a cis guide RNA.

RNA editing in trypanosomes is a post-transcriptional process responsible for correcting the coding sequences of many mitochondrial mRNAs. Uridines are specifically added or deleted from mRNA by an enzymatic cascade in which a pre-edited mRNA is specifically cleaved, uridines are added or removed, and the corrected mRNA is ligated. The process is directed by RNA molecules, termed guide RNAs (gRNA). The ability of this class of small, noncoding RNA to function in RNA editing is essential for these organisms. Typically, gRNAs are transcribed independent of the their cognate mRNA and anneal to form a binary RNA complex . An exception for this process may be cytochrome oxidase subunit II (COII) mRNA since a gene encoding a trans acting gRNA has not been identified. Using an in vitro editing assay we find that the 3' UTR of COII, indeed, functions as a guide for both the site and number of uridines added to the coding region of the COII mRNA. We further show that the guiding sequence within the COII 3' UTR can only function in COII editing when contiguous with the editing substrate, indicating that the 3' UTR of COII lacks sequence or structure information necessary to function as a trans-acting gRNA. While other RNAs have been shown to "guide" RNA processing reactions, our discovery that the COII 3' UTR directs editing of its cognate mRNA in cis, is a unique function for a 3' UTR. The findings described here have led us to propose a new model for the evolution of gRNAs in kinetoplastids.

3' Untranslated Regions↗