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Aerobic exercise increases oxidant stress and leads to apoptosis of mouse intestinal lymphocytes (IL). The purpose of this study was to determine whether freewheel running prevents IL loss 24 h after a bout of strenuous exercise. Mice were randomly assigned to in-cage running wheels with 24 h access (WR) or individual cages without running wheels (NR) for 4 months. WR mice accumulated 364 km over 4 months and had higher cytochrome oxidase activity in the plantaris (p < .05), indicative of training. Total intestinal, CD3alphabeta, CD3gammadelta, CD8alpha, and CD8beta lymphocytes and intracellular glutathione were significantly lower in WR and NR mice 24 h post-exercise. The number of CD4 IL decreased 24 h after exercise in NR (p < .01) but not in WR mice relative to their respective no exercise controls. Thus, freewheel running in mice for 4 months prevented CD4 IL loss after acute exercise.

Increased ventricular ANF-mRNA is observed as a compensating mechanism in response to many forms of overload including chronic exercise. It is unknown if exercise-induced increases in ANF expression are mediated at the transcriptional level and whether specific cis-element(s) may be involved. To study this, rats were injected with several different ANF reporter plasmids. Following recovery, the animals were made to swim for 60 minutes per day. One week of swim training significantly increased plantaris muscle cytochrome oxidase activity and left ventricular ANF-mRNA levels. Exercise training significantly increased ANF reporter expression. We have previously demonstrated that a single cis-element (POE) mediates pressure overload activation of ANF expression during developing hypertrophy. Site-specific mutation of the POE cis-element (-495/-489) within the ANF promoter also ablated exercise-induced increases in reporter expression indicating that the POE site was critical. Further, exercise increase the expression of a POE-heterologous construct, indicating that the POE cis-element alone was sufficient for exercise induced activation. Gel mobility shift analysis, using the POE cis-element as a probe, determined that a single complex was formed and that training did not influence the DNA-protein binding pattern. These results indicate that increased ANF expression is mediated at the transcriptional level as an early adaptation to chronic exercise. The same cis-element that mediated pressure-overload activation of ANF reporter expression was also found to be critical and sufficient to mediate exercise-induced increases in ANF reporter expression. These findings suggest that there may be some overlap in the response to physiological and pathological forms of overload during the phase of developing hypertrophy.

Freezing lesions to the developing cortical plate of rodents results in a focal malformation resembling human 4- layered microgyria, and this malformation has been shown to result in local and widespread disruptions of neuronal architecture, connectivity, and physiology. Because we had previously demonstrated that microgyria caused disruptions in callosal connections, we hypothesized that freeze lesions to the postero-medial barrel sub-field (PMBSF) in one hemi- sphere would affect the organization of this barrel field contralaterally. We placed freeze lesions in the presumptive PMBSF of neonatal rats and, in adulthood, assessed the architecture of the ipsilateral and contralateral barrel fields. Malformations in the PMBSF resulted in a substantial decrease in the number of barrels as identified by cytochrome oxidase activity. More importantly, we found an increase in the total area of the contralateral PMBSF, although there was no difference in individual barrel cross-sectional areas, indicating an increase in the area of inter-barrel septae. This increase in the septal area of the contralateral PMBSF is consistent with changes in callosal and/or thalamic connectivity in the contralateral hemisphere. These results are another example of both local and widespread disruption of connectional architecture following induction of focal microgyria. q 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.

This paper evaluates the changes of cytochrome oxidase (CO) activity that take place in the suprachiasmatic nucleus (SCN) during the light-dark cycle. CO is a mitochondrial energy-generating enzyme used as a marker of neural oxidative metabolism. We measured CO activity using quantitative histochemistry calibrated with brain tissue standards and a computerized analysis image system. The results indicate that the CO enzyme activity changes on the basis of a circadian pattern, with the higher levels during the light phase (P0.0001). These changes are detected over a period of hours, in accordance with other studies on the possible short-term regulation of CO activity in the nervous system. It is, therefore, possible to apply this methodology to the study of the SCN and other brain areas which show functional rhythmicity.

Fronto-opercular and insular cortices of Japanese macaques were histochemically stained for cytochrome oxidase activity. The laminated pattern of enzyme activity differed in the different cytoarchitectonic areas. Area G, the presumed primary gustatory area, as well as area 3 were prominent because the third stripe, corresponding to the termination layers of the specific thalamocortical projection in these areas was very dark and thick in comparison with that from the neighboring areas. This approach provides a convenient histological aid for identification of area G.

The cytochrome oxidase activity of circulating leucocytes was investigated by means of a semiquantitative method using blood films. A significant decrease in activity was found in male rats, cattle and sheep that had been deprived of copper. During copper depletion, leucocyte cytochrome oxidase activity declined more slowly than did plasma copper concentration and plasma ferroxidase I activity, and so was less sensitive as a guide to the copper status of the animal.

Summary Frontal sections from the rabbit olfactory bulb (OB) were stained for cytochrome oxidase (CO) activity. This technique revealed that the OB showed a concentric banding pattern of oxidative enzyme activity. This striped pattern was bilaterally symmetric and continued throughout the OB from rostral to caudal. The most densely stained band was in the middle of the external plexiform layer (EPL). Therefore, regarding differences in degrees of oxidative enzyme activity, the EPL can be divided into three sublayers. In the glomerular layer, individual glomeruli stained as isolated patches and the internal plexiform layer (IPL) was also stained. In contrast to the OB the accessory olfactory bulb stained lightly for CO activity, and did not show a concentric pattern.

The effects were studied of methyl isocyanate (MIC) and its thermally degraded products (dMIC) on rat brain cytochrome oxidase activity. Pure MIC did not inhibit brain cytochrome oxidase activity. A significant inhibition of brain cytochrome oxidase activity by dMIC was observed both in vivo and in vitro. The presence of cyanide in pyrolysed products of MIC has also been confirmed by chemical methods.

Summary Following red light irradiation of excised zucchini hypocotyl hooks and tissue homogenization in a buffer containing EDTA about 50% of the cellular phytochrome became sedimentable into a crude mitochondrial fraction. Upon further analysis by sucrose gradient centrifugation, phytochrome in the crude mitochondrial fraction was distributed near the mitochondrial region and the gradient/sample interface of the gradient. Neither of these two areas of phytochrome activity exactly coincided with the RNA-containing material. Treatment of the crude mitochondrial fraction with Triton ×100 solubilized the phytochrome, RNA-containing material, and the NADPH cytochrome c reductase activity but not the cytochrome c oxidase activity. Under this condition phytochrome and RNA-containing material still appeared to sediment separately. When homogenized in the presence of EDTA, phytochrome and RNA-containing material remaining in the post-mitochondrial supernatant were eluted as separate zones from Sepharose 4B/CL columns, phytochrome being the slower eluting material. When red light irradiated hooks were homogenized in a buffer containing Mg ++ , phytochrome in the post-mitochondrial supernatant was found to elute faster than the RNA-containing material. But in neither case were phytochrome and RNA-containing material eluted together. Pretreatment of red lightirradiated hooks with glutaral-dehyde resulted in most of the phytochrome in the crude mitochondrial fraction to sediment apparently with the cytochrome oxidase activity and RNA containing material. Triton ×100 treatment of the crude mitochondrial fraction did not appear to solubilize the phytochrome and RNA-containing material but caused them to sediment separately. They banded with the cytochrome c oxidase activity on prolonged centrifugation. In post-mitochondrial supernatant from glutaraldehyde-treated tissues, phytochrome and RNA-containing material were eluted from Sepharose columns as separate zones, phytochrome being the slower component.

(1) Phosphine (0.1 mM) when added to preparations of mitochondria from susceptible or resistant R. dominica inhibits the cytochrome c oxidase activity by up to 90%. (2) Cytochrome oxidase activity in mitochondria or crude homogenates of R. dominica of either strain which have been treated in vivo with phosphine shows little effect. (3) Possible mechanisms of resistance are described in the light of this and other evidence.

The laminar distributions of cytochrome oxidase activity and vascularization of the occipital cortex of animals made micrencephalic by exposure to MAM Ac on E14 or E16 were examined and compared to control animals. Despite severe disruptions of the normal cytoarchitecture in MAM Ac exposed animals, the laminar distributions of vascular profiles, branch points and cytochrome oxidase activity in micrencephalic animals were similar to those in control animals.

Cytochrome oxidase histochemistry revealed patchy patterns of the enzyme activity in transverse sections through the caudal part of the ventral subnucleus of the principal sensory trigeminal nucleus, interpolar spinal trigeminal nucleus, and layer IV of the caudal spinal trigeminal nucleus in the cat. By the transganglionic transport method of horseradish peroxidase, the patterns were indicated to replicate the spatial array of the facial vibrissae.

The identification of different anatomical regions of the periaqueductal gray matter of rats was addressed in the present study by using the histochemical staining for the mitochondrial enzyme cytochrome oxidase. At caudal and middle levels, cytochrome oxidase histochemistry clearly demonstrates the existence of four subdivisions: dorsal, dorsolateral, ventrolateral and medial, whereas in sections from the rostral periaqueductal gray matter only two concentric bands are identifiable on the basis of the degree of cytochrome oxidase activity.

Lesions were found in the pancreas of clinically normal cattle of low copper status. In comparison with the pancreas of cattle with normal hepatic copper reserves, the abnormalities were an increase in the dry matter content and reduction in the concentrations of protein and copper in the wet tissue. Cytochrome oxidase activity and protein-to-RNA ratio were also reduced. Histologically, there were defects in acinar basement membranes, splitting and disorganization of acini, cellular atrophy and dissociation, and stromal proliferation. The pancreatic ductular system did not show atrophy or disorganization.

Young Friesian steers were fed for nine months on copper supplemented or depleted diets. Immediately after they had been killed, tissues were taken from the myocardium for examination by electron microscopy. Copper deficiency was associated with extensive changes in myofibrillar and mitochondrial morphology and distribution. It is suggested that the ultra-structural changes in the myocardium were specifically attributable to copper deficiency, the most likely underlying biochemical defect being lost of cytochrome oxidase activity.

Rat heart muscle was spectrophotometrically assayed for succinic dehydrogenase and cytochrome oxidase activity to determine the extent of cryoprotection afforded by DMSO-glycerol solutions. Activities of these two enzymes were greater in the frozen untreated heart than in the normal heart. It was also found that the cryoprotectant without freezing tended to increase the activity of succinic dehydrogenase while cytochrome oxidase activity decreased.

Prolonged isoproterenol administration increases the monoamine oxidase (MAO) but not the cytochrome oxidase activity of rat salivary glands. This increase in MAO can be blocked by prior administration of propranolol or actinomycin D, but is not diminished by ganglionectomy or duct ligation. Three hours after isoproterenol an increase in MAO activity is detectable, but is not significantly inhibited by propranolol. No increase in cardiac MAO concentration was observed. It is suggested that the increased MAO activity may be related to increased enzyme protein in the stimulated secretory cells.

Summary Using the DAB method, cytochrome oxidase activity could be detected in isolated rat thymus nuclei, confined both to membranes of the nuclear envelope and intranuclearly in the euchromatic regions, mostly in the form of granules and sometimes of bodies. Detergent-treated nuclei show the intranuclear deposits almost exclusively.

A simple procedure for the purification of cytochrome oxidase from mitochondria of higher plants is described. The enzyme from wheat, beans, cauliflower and sweet potato was purified 20-, 15-, 20- and 11-fold respectively. Differences in specific activity and Km were observed for the different species. The qualitative differences in cytochrome oxidase activity may be due to the interspecific genetic differences.

The glycogen content of the liver of the frog ( Rana pipiens ) was determined between 60 and 70 hours after single intraperitoneal injections of 2.7 to 86.4 μmoles of CCl 4 per gram body wt of CCl 4 . An inverse relationship existed between the concentration of CCl 4 injected and the quantity of glycogen that the liver contained. No differences were found between the glycogen content of animals sacrificed at 60 hours after injection of CCl 4 , and those sacrificed at 70 hours with the same concentration of CCl 4 . As the liver glycogen became depleted, a corresponding increase occurred in the blood-glucose level. The limit of tolerance to CCl 4 approached 172.8 μmoles per gram body wt. This concentration was greater than LD 99 . Carbon tetrachloride at a concentration of 6.0 μmoles per gram body wt produced a 17% increase in the cytochrome-oxidase activity.

Ground beef and pork loin irradiated with 2.79 or 5.58 million rads and subsequently stored at either 70° F or 100° F have been fed ad libitum to rats for 8–12 weeks as 35% of the dry solids of the diet. The remaining 65% of the diet was a nonirradiated, nutritionally adequate semisynthetic ration. Comparable nonirradiated foods were used as controls. Covariance analysis of the initial weights, weight gains, and caloric consumption data was performed to determine low levels of toxic products in the irradiated foods. No indications of toxicity were obtained with any of the diets. Six liver and blood enzymes were assayed as indexes of subtle changes in metabolism due to consumption of irradiated foods. Only liver cytochrome oxidase activity exhibited any alteration. This enzyme was increased with increasing radiation dosage. Cooking of the 2.79 megarad-irradiated beef enhanced the increase, but cooking of the control or 5.58-megarad products did not cause further change. The implications of these results are discussed.

The extent of inhibition of cytochrome oxidase activity by cyanide depends upon the method of assay. It has been confirmed that this inhibition is "readily reversible" with the use of the standard manometric technique. However, in the spectrophotometric assay only about 10% of the control activity is observed after the cyanide is removed by physical methods. The polarographic assay yields intermediate results. On the other hand, the polarographic assay shows a complete reversal of inhibition when the cyanide-treated heart muscle preparation is first incubated under reducing conditions. The tetrachlorohydroquinone oxidase activity of the heart muscle preparation is also decreased by cyanide treatment. The spectra of the purified cytochrome oxidase treated with cyanide are similar to those of cytochrome a. It is likely that there are two sites for the interaction of cytochrome oxidase and cyanide: the heme a iron and a second noncovalent locus. The nature of this second site s discussed.

The cytochrome oxidase activity of isolated mitochondria of unfertilized and fertilized eggs of Paracentrotus lividus has been studied. It has been found that whereas in the mitochondria of unfertilized eggs saturation is reached at a cytochrome c concentration of 3.28 × 10−5 M, in those of fertilized eggs this occurs at a concentration of 5.74 × 10−5 M. It is shown that upon fertilization an increase of about 30 per cent of the activity of cytochrome oxidase takes place and no further changes appear to occur until blastula stage.

Normal male BDF1 mice, maintained on a toxic level (40 mg/kg/day) of 1-β-d-arabinofuranosylcytosine (ara-C) exhibited marked alterations in the cytochrome oxidase activity of spleen and marrow during treatment. Splenic cytochrome oxidase activity was elevated initially, and then decreased during treatment for 60 days with a well-tolerated level of ara-C (20 mg/kg/day). Toxicity was also evident fromprogressively increasing spleen to body weight ratios during treatment and the splenic cytochrome oxidase activity varied inversely with splenomegaly. Similar phenomena were observed in mice inoculated with Leukemia L1210 during treatment with ara-C. The comparative absence of hepatotoxicity was suggested by the relatively normal hepatic cytochrome oxidase activities observed during treatment with the optimal level of ara-C (20 mg/kg/day). In studies conducted in vitro, ara-C did not inhibit normal splenic cytochrome oxidase thereby indicating that the observed effects of ara-C were an indirect consequence of treatment. The assay for cytochrome oxidaseis useful for evaluation of toxicity during treatment with ara-C and for the formulation of regimens which have less adverse effects on the host.

1. 1. Cytochrome oxidase activity shows a precise exponential relationship to total O 2 consumption among various vertebrate species with different O 2 , consumptions. A similar relationship between cytochrome oxidase activity and organ O 2 consumption has been demonstrated in a single species (rabbit). 2. 2. These findings suggest that cytochrome oxidase activity may represent a quantitative biochemical marker for functional mitochondrial mass. 3. 3. The regulation of cytochrome oxidase activity may represent a long-term regulatory mechanism involving mitochondrial O 2 uptake in different species and different tissues.

1. 1. Cytochrome oxidase activity, determined over the range 10–40°C was higher at all assay temperatures in brain, gill and muscle homogenates of 10°C-acclimated goldfish than 30°C-acclimated fish, but was only higher in liver homogenates when assayed at 30 and 40°C. 2. 2. Thermal compensation of gill cytochrome oxidase did not appear to involve a significant increase in the mitochondrial content of the cells. 3. 3. Cytochrome oxidase, succinate-cytochrome-c reductase and NADH-cytochrome-c reductase activities were all higher in gill mitochondrial preparations from cold-acclimated fish. 4. 4. The concentration of cytochrome-c1 was reduced in gill mitochondria of cold- compared to warm-acclimated goldfish. However, the concentrations of cytochromes-aa3, -b and -c were similar in these two groups. 5. 5. It nis suggestes that mitochondrial electron transport systems compensate to the degree necessary to meet changing ATP demands of goldfish exposed to new environmental temperatures.

The myeloperoxidase content of the normal human leucocyte has been established as greater than 5% of the dry weight, which is contrary to the often quoted value of 1–2%. The conditions for the preparation of a new cellular particulate in which the peroxidase activity is localized is described; it is heavier than mitochondria, and contains at least 10–30 times more peroxidase than the particulate fractions carrying the cytochrome oxidase activity.

Phenolic substances, which become oxidized during the preparation of mitochondria from lettuce seeds, co-precipitate with mitochondria during their isolation. Phenolase present in the mitochondria maintains these phenolics in the oxidized state during the assay of cytochrome oxidase and as a result the activity of the latter enzyme is depressed. The depression is removed by inhibitors of phenolase. This mechanism may also effect cytochrome oxidase activity in vivo .