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Department of Biochemistry and Molecular Biology, University of Minnesota, Duluth, MN 55812
2To whom correspondence should be addressed. E-mail: jprohask{at}d.umn.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • copper deficiency • rat brain • cytochrome c • apoptosis • Bcl-2
Copper is an essential element for all developing mammals. In mice, for example, the absence of the plasma membrane copper transporter, Ctr1, leads to growth and developmental defects, ultimately resulting in death in utero (1,2). Similarly, mouse pups deficient in the copper metallochaperone, Atox1, display profound growth retardation and congenital malformations, and exhibit a 45% mortality rate before the time of weaning compared with wild-type mice (3). In humans, the X-linked copper deficiency disorder, Menkes syndrome, is another example of copper’s essentiality. This disorder, caused by the loss of a P-type ATPase responsible for copper efflux from cells, leads to copper accumulation in gut epithelial cells and a deficiency in all other tissues. Menkes patients are characterized by hypothermia, hypopigmentation, loose skin, weakened arteries and marked neurodegeneration usually resulting in death at the age of 3–4 y (4).
The effects of perinatal dietary copper deficiency on the development of the central nervous system (CNS)2 have been extensively documented in laboratory animals as well as in humans (5). Dietary copper deficiency studied in laboratory rodents showed severe developmental and morphological abnormalities of the brain (6). In 1969 Carlton and Kelly reported gross neural focal lesions in the occipital and parietal parts of the cerebral cortex as well as pathoanatomic alterations in the corpus striatum of copper-deficient rats (7).
Many of the deleterious effects of copper deficiency have been ascribed to the loss of cuproenzyme activity. Copper is key to the function of enzymes such as Cu, Zn-superoxide dismutase (SOD), cytochrome c oxidase (CCO), dopamine-ß-monooxygenase (DBM) and peptidylglycine-
-amidating monooxygenase (PAM). Copper’s varied biological roles in the CNS range from defense from free radical damage to oxidative phosphorylation, neurotransmission and neuropeptide maturation (8).
Electron microscopic examination of the brain tissue of copper-deficient rats reveals enlarged mitochondria (9). Copper is a cofactor of CCO, the complex IV of the electron transport chain essential for the maintenance of cellular energy metabolism through oxidative phophorylation. In copper-deficient rats, CCO activity can be reduced by as much as 80% (9). Analysis of brain tissue from male brindled mutant mice (Mobr/y), a genetic model of copper deficiency, has shown a nearly 50% reduction in cellular ATP concentration (10). Other studies have also shown abnormally swollen mitochondria in both the normal and degenerating neurons of mice (11).
Mitochondrial dysfunction plays an essential role in the initiation of and the commitment to apoptosis or programmed cell death (12). Cytochrome c is a normal component of the electron transport chain found in the intermembrane space tethered to inner membranes. However, during apoptosis mitochondria release cytochrome c into the cytoplasm where it binds adapter protein Apaf-1 and pro-caspase-9. Subsequently, this protein complex leads to the activation of "executioner" caspases such as caspase-3 (13). Caspase-3, among other activated proteases, feeds the proteolytic cascade leading to cellular decomposition (14). In contrast to the proapoptotic role of cytochrome c release, another mitochondrial membrane protein, Bcl-2, plays a key role in preventing the initiation of apoptosis (15).
Others have noted that brain tissue from brindled mice has enhanced cytochrome c release into the cytosol and a striking reduction of mitochondrial Bcl-2, suggestive of apoptosis in this model of copper deficiency (10). Based on mitochondrial anomalies in morphology and lower CCO activity in the rat brain, we tested the hypothesis that apoptosis occurs in the brain following dietary copper deficiency using several markers including cytochrome c release and Bcl-2 content.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plug-positive Holtzman rats were purchased commercially (Harlan Sprague-Dawley, Indianapolis, IN). Dietary treatment was of two types: copper-deficient and copper-adequate. Rats were fed a copper-deficient diet (TD-80388; Teklad Laboratories, Madison, WI) and drank either purified or copper-supplemented water. The purified diet was similar to the AIN-76A diet and contained the following major components (g/kg diet): sucrose, 500; casein, 200; cornstarch, 150; corn oil, 50; cellulose, 50; modified AIN-76 mineral mix, 35; AIN-76A vitamin mix, 10; DL-methionine, 3; choline bitartrate, 2; and ethoxyquin 0.01 (16,17). Cupric carbonate was omitted from the AIN-76 mineral mix. The purified diet contained 0.31 mg/kg of Cu and 47 mg/kg of Fe by chemical analysis. Copper-supplemented drinking water, made by adding CuSO4, contained 20 mg/L of Cu. After weaning, the offspring of dams from the two treatment groups received the same dietary treatment as the dams. Rats were given free access to food and drinking water. All rats were maintained at 24°C with 55% relative humidity on a 12-h light/dark cycle (lights on 0700–1900 h). All protocols were formally approved by the University of Minnesota Animal Care Committee.
Pregnant dams were fed their respective diets 7 d into gestation. Two days after birth (P2) each litter was adjusted to 10 pups and were weaned at P20. A total of 8 litters (4 copper adequate and 4 copper deficient) were sampled.
Sample collection.
Male rat pups were sampled at age P12–13 and P24–25. After decapitation, blood samples were drawn from the torso into heparinized microhematocrit tubes. An aliquot for hemoglobin analysis was also taken. After dissection, plasma and packed red blood cells were obtained by centrifugation at 13,000 x g for 10 min. The brain was promptly removed, placed on an ice-cooled watch glass, rinsed with cold deionized water and weighed. The cerebellum, medulla oblongata/pons and hypothalamus were dissected and the remainder of the brain (cortex) was cut medially; one-half was used for metal analysis and one-half for biochemical and immunoblot analyses. Cortices and cerebella were homogenized with 4 volumes of 0.32 mol/L sucrose solution, pH 7.0, with a Potter-Elvehjem type homogenizer (4 full strokes). The homogenate was further diluted with sucrose (total 100x) for enzymatic analysis. A portion of the liver was removed, rinsed with deionized water, weighed and processed for metal analysis. The whole heart was also taken, rinsed with deionized water, weighed and stored at -80°C for later analysis.
Histochemistry.
After being deeply anesthetized with halothane, the brains of P12 and P25 male rat pups were fixed by transcardial perfusion. Briefly, the brain was flushed of blood with heparinized PBS, pH 7.4, and then fixed over a period of 10–12 min with 3.7% (37 g/L) formaldehyde in PBS, pH 7.4. The brains were then removed, postfixed, embedded in paraffin and sectioned into 8-µm-thick serial coronal slices. The morphological characteristics were determined using light microscopy with hematoxylin-eosin staining. The number of brain cells exhibiting DNA fragmentation in copper-adequate and copper-deficient rats was evaluated in situ by means of the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) technique. TUNEL staining was performed using the ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Serologicals, Norcross, GA) according to the manufacturer’s instructions. TUNEL-positive cells were counted in 15 separate fields (0.16 mm2) per tissue section under light microscope (40x magnification) by an observer blind to diet group. The areas observed corresponded to neocortex and all layers of the cerebellar cortex.
Chemical analyses.
Portions of the liver and cerebral cortex were wet-digested with 4.0 mL of concentrated HNO3 (TraceMetal grade; Fisher Scientific, Pittsburgh, PA) and residue was brought to 4.0 mL with 0.1 mol/L HNO3. Samples were analyzed for copper and iron content by flame atomic absorption spectroscopy (Model 2380; Perkin-Elmer, Norwalk, CT). Protein content of tissue samples was determined using a modified version of the Lowry method with bovine albumin as a reference (18). Hemoglobin was determined specrophotometrically as metcyanohemoglobin (19).
Enzyme assays.
Activites of cuproenzymes were determined by protocols detailed elsewhere (19). Plasma ceruloplasmin diamine oxidase activity was determined by using o-dianisidine as substrate at pH 5.0. The activity of Cu, Zn-SOD was measured spectrophotometrically by determining the inhibition of pyrogallol autooxidation at pH 8.2 at 320 nm. CCO activity of fresh tissue homogenates (100x) treated with 0.1% Triton X-100 was measured spectrophotometrically (Beckman DU-640) by monitoring the loss of ferrocytochrome c (initial concentration 50 µmol/L) at 550 nm.
The caspase activity of brain samples was determined using the Caspase-3 Colorimetric Assay Kit (Assay Designs, Ann Arbor, MI). Aliquots of cortex and cerebellum, original 5x homogenates, were mixed with 9 volumes of assay buffer, provided by the manufacturer, containing 15 g/L of fresh dithiothreitol. Samples were then vortexed, rehomogenized and centrifuged for 20 min at 25,000 x g at 4°C. Supernatants were processed according to the manufacturer’s instructions.
Western immunoblot analysis.
Western immunoblot analysis was performed on the cytosolic and total protein extracts from brain and heart tissues. To obtain cytosolic protein, tissues were minced and gently homogenized with 4 volumes of ice-cold 0.32 mol/L sucrose, pH 7.0, using a chilled Potter-Elvehjem teflon-on-glass homogenizer. Four full strokes were used for homogenization to maintain subcellular organelle integrity. Protease inhibitors (Protease Inhibitor Cocktail; Sigma, St. Louis, MO) were added to the samples. Samples where then centrifuged at 14,000 x g for 15 min at 4°C. Supernatants, containing predominantly soluble proteins, were stored at -80°C until later analysis. Total protein extracts were prepared by treating an aliquot of tissue homogenate with 0.5% (5 g/L) Triton X-100. Samples were then further homogenized, vortexed, sonicated for 10 s and centrifuged at 14,000 x g for 15 min at 4°C. Supernatants, containing both soluble and membrane proteins, were stored at -80°C until later analysis.
Western blotting analysis was performed by size fractionation of proteins on 15% SDS-PAGE gels and electroblot transferring to 0.2-µm nitrocellulose membranes (Protran; Schleicher & Schuell, Keene, NH). Membranes were stained with Ponceau S solution (Sigma) to ensure equal loading of the protein, then washed for use in immunoblotting as described elsewhere (20). Some membranes were stripped and reprobed after incubation with buffer containing 2-mercaptoethanol and SDS at 55°C.
Specific reagents were used to detect Bcl-2, cytochrome c, copper chaperone for superoxide dismutase (CCS), the 39-kDa peptide of mitochondrial complex I, and mitochondrial voltage-dependent anion channel (VDAC). Monoclonal anti-Bcl-2 antibody (PC333; Oncogene Research Products, San Diego, CA) was used at a 1:300 dilution. Polyclonal anti-cytochrome c antibody (cs-7159, Santa Cruz Biotechnology, Santa, Cruz CA) was used at a 1:500 dilution. CCS rabbit antiserum was produced commercially (Sigma Genosys, The Woodlands, TX) and used at 1:1000 dilution. The design of the peptide antigen and characterization of the rabbit anti-CCS serum were described earlier (21). Monoclonal antimitochondrial complex I antibody (A-21344; Molecular Probes, Eugene, OR) was used at 0.5 mg/L concentration to detect mitochondrial complex I. Polyclonal anti-VDAC antibody (sc-8829, Santa Cruz, Santa Cruz, CA) was used at a 1:700 dilution. Horse heart cytochrome c (Sigma) was used as a positive control and molecular weight standard. All secondary species specific antibodies were diluted 1:10,000 except for cytochrome c which was diluted 1:5,000. SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL) was used to detect selected proteins. Chemiluminescence was captured using high speed blue X-ray film (Lake Superior X Ray, Duluth, MN) and densitometry was carried out using the FluorChem system (Alpha Innotech, San Leandro, CA). The size of the immunoreactive bands was estimated from regression analysis using standard peptides (Bio-Rad, Hercules, CA).
Statistical analysis.
Means ± SD or SEM were calculated. The Student unpaired two-tailed t test was used to compare data between the two dietary treatments ( = 0.05). Factorial ANOVA was used to evaluate diet and age for cytochrome c data. Interactions were evaluated by the Fisher PLSD test.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male pups from two dietary treatments, copper-adequate and copper-deficient, were analyzed for copper status at two developmental stages (suckling and postweanling), 12–13 and 24–25 d-of-age. Thirteen days after birth (P13), copper-deficient rat pups weighed less than their copper-adequate counterparts, a characteristic that was more pronounced at P24 (Table 1). At both ages, plasma ceruloplasmin diamine oxidase activity was barely detectable in the copper-deficient rats compared with the copper-adequate controls (Table 2). Brain CCO activity was lower in the copper-deficient rats than in the copper-adequate rats at both P13 and P24 (Table 2). These data suggest that the dietary model was successful at producing rats of two distinct copper states: deficient and adequate.
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Cytochrome c.
To evaluate apoptosis, the brains of P13 and P24 rats were analyzed by Western blotting for soluble cytochrome c. Analysis showed an immunoreactive band estimated to be 12 kDa, the approximate molecular weight of cytochrome c, for both brain regions (Fig. 1). However, densitometry analysis of the immunoreactive cytochrome c bands from these same blots showed no difference between the two treatment groups. The copper status of the rat brain supernatants was evaluated by stripping the Western blot membranes and immunoblotting for CCS. This predominantly cytosolic protein has previously been shown to be upregulated in copper-deficient rats (22,23). Higher immunoreactivity in the copper-deficient samples (P < 0.05) confirmed that supernatants from the cortices and cerebella of copper-deficient rats had altered copper status. In the cortex, CCS protein from copper-deficient rats was higher, 36.3% at P13 and 68.1% at P24, compared with controls. In the cerebellum, CCS in P13 copper-deficient rats was higher by 41.3 and 48.5% at P25.
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Both mitochondrial and total protein extract immunoblots used previously for cytochrome c quantification were reprobed for Bcl-2, an antiapoptotic protein (Figs. 2, 3). No change in Bcl-2 content was observed between copper-deficient and copper-adequate samples. This was true for total protein extracts from the cortices and cerebella of both P13 and P24 rats (Fig. 3) and of purified mitochondria (Fig. 2).
The conflicting results of total cytochrome c and Bcl-2 prompted us to use another measure of neuronal apoptosis by measuring caspase-3 activity in the cortex and cerebellum. Caspase-3 activity at P13 was higher (59.8%) in the cerebella of copper-deficient rats compared with controls (Fig. 4). However, we failed to detect differences in the cortices between the two treatment groups at P13. Brain tissue samples from P24 rats of both dietary treatments showed no detectable caspase-3 activity in either brain region (results not shown).
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To verify TUNEL and caspase-3 activity results, cytochrome c content was reevaluated at the two ages studied. To eliminate any inconsistencies that occurred in the Western blotting process it was necessary to compare both P12 and P25 cortex or cerebellar extracts on a single gel transferred onto to same membrane (Fig. 6). As analyzed by 2 x 2 factorial ANOVA, cortex cytochrome c was higher in copper-deficient extracts at both ages (effects of both diet and age, no interaction) (19% at P12 and 30% at P25, respectively) (Fig. 6). In the cerebellum there was also an effect of both diet and age and an interaction. Cytochrome c content was also elevated in the P25 copper-deficient rats (76%). In the P12 rats there was a trend for higher cytochrome c content (P = 0.0804), with a mean 38% higher level. Thus, elevated cytochrome c data seemed unrelated to other apoptosis data.
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The relative quantities of other mitochondrial markers were assessed in the brain of P24 rats using Western immunoblots. VDAC protein and 39-kDa subunit of complex I of the mitochondrial electron transport chain were measured. Compared with controls, the P24 copper-deficient rat cortices displayed 28% higher levels of complex I peptide and 46% higher levels of VDAC (Fig. 7). Immunoblots of purified rat brain mitochondrial extracts were also analyzed for these two mitochondrial markers. Western blots revealed a 25% increase in complex I and a 54% increase in VDAC (Fig. 2).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We evaluated the possibility that mitochondrial-dependent neuronal apoptosis accompanied perinatal dietary copper deficiency in rats. Four independent measures (cytochrome c, Bcl-2, caspase 3 activity and TUNEL staining) failed to demonstrate that specific apoptosis was associated with copper deficiency in our perinatal model. This contrasts with other studies using a genetic model, brindled mice, that exhibits signs of copper deficiency due to loss of copper efflux transporter, ATP7A (10). These studies reported marked release of cytochrome c into cytosol, a classic apoptotic characteristic. Also, TUNEL staining was increased in brindled mouse brain and in the brain of a related mutant, macular mutant mouse (26). Together they support apoptosis as a mechanism to explain neuronal cell death. Perhaps species differences (mouse versus rat) or the earlier onset of copper deficiency (throughout gestation in the mouse mutants) accounts for our failure to detect apoptosis in rat brain.
Our results indicate that changes in apoptotic markers correlated better with age than with degree of copper deficiency. For example, we found no detectable difference in cytosolic cytochrome c content in rat brains of either developmental age despite increasing copper deficiency. However, we did detect a robust enhancement of total cytochrome c content in copper-deficient brains. At both developmental ages copper-deficient rat brains exhibited a greater total cytochrome c content than controls. As the rats aged, the elevation in total cytochrome c content in copper-deficient compared to copper-adequate cerebella increased by 38%, from 38% higher at P13 to 76% higher at P24. This age effect was reflected by altered SOD activity. At P13 no diet treatment difference in SOD activity was detected; however, at P24 there was a 33% decrease of SOD activity in both brain regions of copper-deficient rats suggesting, along with greater reductions in cortex copper concentrations, a more severe copper deficiency at P24. In contrast, as the rats aged, apoptotic markers caspase-3 activity and TUNEL staining decreased. At P12–P13 cerebellar caspase-3 activity and TUNEL staining in copper-deficient pups was elevated. At P24–25, TUNEL staining in both control and copper-deficient cerebella decreased from P12 levels and there was no detectable caspase-3 activity. Higher caspase-3 activity and TUNEL staining at P12–13 most likely correlates with apoptosis associated with brain remodeling in the developing brain rather than a dietary copper deficiency effect (27). Although there was a dietary effect seen in cerebellar caspase-3 activity at P13 and TUNNEL staining at both P12 and P25, these effects are more likely related to a delayed development of the cerebellum of copper-deficient rats (28,29). We conclude that the increase in total cytochrome c is due to reasons other than apoptosis.
Bcl-2, an antiapoptotic mitochondrial protein, was unaltered in concentration in both brain regions following dietary copper deficiency at both the developmental stages investigated. This contrasts with results obtained by Rossi et al. who showed that copper deficiency in the brindled mouse model was associated with a reduction in Bcl-2 content in the brains of P14 pups (10). However, in contrast to brain, copper-deficient heart tissue of P24 rats showed the reduction of Bcl-2 levels suggestive of cardiac apoptosis and validation of the ability to detect apoptotic changes, if present. Our results in the hypertrophic rat heart are consistent with data on the well established apoptotic hypertrophic heart of mice following dietary copper deficiency described by Kang et al. (30). Bcl-2 levels have been shown to be a key factor in cardiocyte commitment to apoptosis. Past studies have determined overexpression of Bcl-2 to attenuate apoptosis and protect against myocardial ischemia-repefusion injury in transgenic mice (31). Our results showing lower Bcl-2 content in copper-deficient rat heart are therefore consistent with observations of apoptosis in copper-deficient mouse heart based on TUNEL staining (30).
Though unrelated to apoptosis, one notable result of the current study with copper deficiency is the increase in brain total cytochrome c content. There are a number of possible explanations. One hypothesis is enhanced mitochondrial proliferation accompanying dietary copper deficiency. Dietary copper deficiency is known to alter the morphology of mitochondria in the liver, heart and brain (9,32,33). Mitochondrial proliferation and hypertrophy is well documented in the heart of copper-deficient rats and mice (30,34). Thus, the observed increase in cytochrome c in isolated brain mitochondria and detergent extracts of brain tissue may reflect increased mitochondrial membrane mass in copper-deficient brain. Supporting this hypothesis, we detected increased levels of other mitochondrial membrane markers, complex I peptide and VDAC. Furthermore, these markers were higher in total protein extracts from the hearts of copper-deficient rats, a tissue with known mitochondrial proliferation. There were some subtle differences between the mitochondrial marker profiles in the heart and brain and in the two brain regions.
Thus, a second hypothesis explaining the elevation of cytochrome c levels following copper deficiency relates to altered metabolism. In dietary copper deficiency brain mitochondria experience more than a 50% reduction in CCO activity yet brain ATP levels and mitochondrial respiration are not altered (9). ATP levels are also not altered in other organs following copper deficiency in rats and mice (35,36). Perhaps the copper-deficient cell compensates. A plausible mechanism compensating for low CCO while striving to maintain normal oxidative phosphorylation and cellular ATP is the enhanced synthesis of cytochrome c. Recent evidence demonstrated cytochrome c upregulation as a means of enhancing mitochondrial respiration without CCO upregulation (37). Measurements in isolated cells and mitochondrial tomographic studies suggest a potentially smaller pool of cytochrome c interacting with cytochrome c oxidase than previously believed. Consequently, increased mitochondrial cytochrome c content, and therefore increased cytochrome c/CCO, may enhance CCO activity and respiration (38). Interestingly, one transcription factor needed for the expresssion of the cytochrome c gene, NRF-1, has been shown by others to be upregulated following dietary copper deficiency in rats (39).
A third hypothesis explaining the elevation in brain cytochrome c relates to a compensatory response due to the enhanced production of reactive oxygen species (ROS) following copper deficiency. The increased cytochrome c seen in brain mitochondria of copper-deficient rats may be a protective mechanism for the compromised SOD antioxidant system and the reason for the absence of lipid peroxidation in brain as previously reported (9). Our results indicate that total cytochrome c levels were highest when SOD activity was lowest. It has been almost 30 y since the first report of lower cytosolic Cu, Zn-SOD activity in brain following dietary copper deficiency (9,28). No evidence of lipid peroxidation, a measure of ROS damage in rat brain, was observed (9). However, others have recently documented lower SOD activity and increased superoxide production in copper-deficient rat embryos cultured in copper-deficient serums (40). Using whole animal studies and ethane gas emission, Lawrence et al. have convincingly shown that copper-deficient rats demonstrate enhanced lipid peroxidation even prior to carbon tetrachloride challenge (41). Thus, it is possible that enhanced ROS are present in brains and hearts of our copper-deficient rats. Interestingly, Sp1, an oxidative stress-inducible antideath transcription factor, binds to the promoter element of the cytochrome c gene (42).
Cytochrome c is thought to be an antioxidant capable of scavenging mitochondrially produced ROS by reacting with superoxide and hydrogen peroxide to produce O2 and H2O, respectively (43–45). In support of this function, cytochrome c depletion augments superoxide and H2O2 generation seven- to eightfold above normal levels (45). Thus, there are several possible mechanisms for elevated cytochrome c levels following copper deprivation.
Our results indicate that dietary copper deficiency does not induce neuronal apoptosis in the rat cortex or cerebellum. It is possible that subtle damage was present but not detected by our methods. Dietary copper deficiency may induce apoptosis of a small number of key neurons, the loss of which could translate into permanent neurological damage and go undetected by tissue caspase-3 activity measurements or TUNEL assays. It is also possible that neuronal apoptosis occurs earlier or later in development than the time chosen for our evaluation of copper-deficient rat brain. The results presented here add to the complexity of the effect of copper deficiency on the CNS by demonstrating increased total cytochrome c content. Further work will be necessary to determine the mechanism for the higher cytochrome c and to determine if this change alters function.
Recovery from perinatal dietary copper deficiency fails to restore copper levels of deficient rats to the same concentration as controls (46). Furthermore, neurological abnormalities such as blunted auditory startle and enhanced foot splay persist even after long-term copper supplementation (47). If inapropos apoptosis is not the mechanism for altered behavior, what could be responsible?
In addition to altered SOD and CCO, the brains of copper-deficient rodents have lower levels of the neurotransmitter, norepinephrine, synthesized by the cuproenzyme, DBM, and higher levels of dopamine, the substrate for DBM (48). Another neurologically important cuproenzyme, PAM, is also altered by copper deficiency (49). Altered DBM and/or PAM function could change the neurochemical milieu during development and lead to behavioral changes without gross lesions leading to the loss of cells. Further research will be required to resolve these issues.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CCO, cytochrome c oxidase; CCS, copper chaperone for superoxide dismutase; CNS, central nervous system; DBM, dopamine-ß-monooxygenase; PAM, peptidylglycine--amidating monooxygenase; ROS, reactive oxygen species; SOD, Cu, Zn-superoxide dismutase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; VDAC, voltage-dependent anion channel.
4 One µg of copper = 15.7 nmol.
Manuscript received 31 July 2003. Initial review completed 12 August 2003. Revision accepted 15 August 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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