CytOx Activity Research Articles

Thermal Inactivation of Ricin Using Infant Formula as a Food Matrix

Ricin is a potent protein toxin found in the seeds of the castor bean plant, Ricinus communis. Ricin specifically and irreversibly inactivates ribosomes, promoting cell death by inhibiting protein synthesis. It is composed of a ribosome-inactivating enzyme (A-chain) linked to a lectin (B-chain) by a single disulfide bond. Several reports indicate that ricin can be detoxified by thermal treatment; however, the conditions required for inactivation are not well characterized. In addition, little information exists on the thermal stability of ricin added to foods. The objective of this work was to determine the effects of heat treatments on the detection and toxicity of ricin added to milk- and soy-based infant formulas. Reconstituted infant formula powders containing 100 mug of ricin/mL were heated at 60-90 degrees C for up to 5 h. The heat-treated formulas were analyzed by ELISA to determine levels of ricin. The residual cytotoxicity of ricin-containing infant formula after heat treatments was determined using RAW264.7 mouse macrophage cells. The ELISA and the cytotoxicity assay indicated that ricin detection and toxicity decreased with increasing heating times and temperatures. Minimal losses in detection and toxicity were found for ricin heated at 60 degrees C for 2 h. The half-lives of ricin cytoxic activity in a milk-based infant formula at 60, 70, 75, 80, 85, and 90 degrees C were >100, 9.8 +/- 0.5, 5.8 +/- 0.9, 5.1 +/- 0.7, 3.1 +/- 0.4, and 1.8 +/- 0.2 min, respectively; the comparable values for a soy-based infant formula were >100, 16 +/- 1.6, 8.7 +/- 1.2, 6.9 +/- 1.1, 3.0 +/- 0.4, and 2.0 +/- 0.3 min. ELISA detection was a good indicator of the cytotoxicity of heat-treated ricin. The results indicate that ricin is a relatively heat stable protein and may remain toxic under some food processing conditions.

77 An interaction between 14-3-3 and ExoS of P. aeruginosa is necessary for ExoS cytoxicity and enzymatic activity

77 An interaction between 14-3-3 and ExoS of P. aeruginosa is necessary for ExoS cytoxicity and enzymatic activity

Review: Tubulin Function, Action of Antitubulin Drugs, and New Drug Development

Anticancer agents that interfere with microtubulin function are in widespread use in man and have a broad spectrum of activity against both hematological malignancies and solid tumors. The mechanisms of actions of these agents have been better defined during the past decade, indicating that there are distinct binding sites for these agents and that they interfere with microtubulin dynamics (growth and shortening of tubules) at low concentrations and only evoke microtubulin aggregation or dissociation at high concentrations. Tubulin has been recently described in the nucleus of cells and in mitochondria. Downstream events from tubulin binding are believed to be critical events for the generation of apoptosis in the malignant cell. The effects of vinca alkaloids and taxanes are distinct, suggesting that the interference with the tubulin cap by high-affinity binding of effective agents is not the only mechanism of cytotoxic effect, and the low-affinity binding of drug, which distorts microtubulin function, may also be important. The epothilones share some of the binding characteristics of the taxanes and are in clinical trials because of cytoxic activity in taxane resistant cells. Tubulin has additional target sites for anticancer drugs including interference with the binding and function of microtubule associated proteins and interference with motor proteins which are essential for the transport of substances within the cell. Because many of these microtubule associated proteins have an ATP binding site, both computer-aided design and combinatorial chemistry techniques can be used to make agents to interfere with their function analogous to imatinib mesylate (Gleevec™). Agents that interfere with the motor protein kinesin are entering clinical trials.

Synthesis and cytotoxic activity of lipophilic sulphonamide derivatives of the benzo[b]thiophene 1,1-dioxide

In the search of new compounds with antineoplastic activity, we have analysed the effect of several structural modifications on the nucleus 6-benzo[b]thiophenesulphonamide 1,1-dioxide on its cytotoxic activity on tumour cells. Lipophilic substituents on the sulphonamide group significantly increased the cytoxic activity measured using a panel of human tumour cell lines. Only slight variations on cytotoxicity were obtained when the sulphonamide group occupied the position 5 of the system. The most active compound was the N-4-methoxyphenyl derivative 15, which showed GI50 values of 1–9 nM against HT-29, CCRF-CEM, K-562 and MEL-AC cells and of 200 nM against HTB-54 cells. Free access to the 3-position of the heterocyclic system seems to be required to obtain cytotoxic derivatives. Derivative 15 was also active at the same level of commercial Doxorubicine against cultured normal human lung fibroblasts.

Natural killer cell-mediated basal and interferon-enhanced cytotoxicity against liver cancer cells is significantly impaired under in vivo oxygen conditions.

Current immunostimulatory treatment protocols of cancer are often met with little success. Several lines of evidence indicate that the tumour microenvironment may impair the cytotoxic activity of natural killer (NK) cells. In this study, the NK cell-mediated killing of liver-derived cells was investigated at oxygen concentrations conform to those present in the human body at physiological and pathological conditions. The in vivo-relevant oxygen concentrations corresponding to 1, 2 and 6% were compared to those of the ambient air (21%) for their effect on the lysis of four liver-derived cell lines and the highly susceptible K562 cells. Exposure to each of the hypoxic conditions had a significantly inhibitory effect on NK cytotoxicity. Treatment with interferon-alpha (IFN-alpha) in hypoxia enhanced the cytotoxic potential of the NK cells less than it enhanced the cytotoxicity at ambient oxygen conditions. In summary, the oxygen tension profoundly affects both the cytoxic activity of NK cells and their activation by IFN-alpha.

Effect of body temperature during exercise on skeletal muscle cytochrome c oxidase content.

This study determined the role of body temperature during exercise on cytochrome-c oxidase (CytOx) activity, a marker of mitochondrial content, and mitochondrial heat shock protein 70 (mtHSP70), which is required for import of nuclear-coded preproteins. Male, 10-wk-old, Sprague-Dawley rats exercised identically for 9 wk in ambient temperatures of 23 degrees C (n = 10), 8 degrees C with wetted fur (n = 8), and 4 degrees C with wetted fur and fan (n = 7). These conditions maintained exercising core temperature (T(c)) at 40.4, 39.2, or 38.0 degrees C (resting temperature), respectively. During weeks 3-9, exercisers ran 5 days/wk up a 6% grade at 20 m/min for 60 min. Animals were housed at 23 degrees C. Gastrocnemius CytOx activity in T(c)=38.0 degrees C (83.5 +/- 5.5 microatoms O x min(-1) x g wet wt(-1)) was greater than all other groups (P < 0.05), exceeding sedentary (n = 7) by 73.2%. T(c) of 40.4 and 39.2 degrees C also were higher than sedentary by 22.4 and 37.4%, respectively (P < 0.05). Quantification of CytOx content verified that the increased activity was due to an increase in protein content. In extensor digitorum longus, a nonactive muscle, CytOx was not elevated in T(c) = 38.0 degrees C. mtHSP70 was significantly elevated in gastrocnemius of T(c) = 38.0 degrees C compared with sedentary (P < 0.05) but was not elevated in extensor digitorum longus (P > 0.05). The data indicate that decreasing exercise T(c) may enhance mitochondrial biogenesis and that mtHSP70 expression is not dependent on temperature.

Perinatal Iron Deficiency Decreases Cytochrome c Oxidase (CytOx) Activity in Selected Regions of Neonatal Rat Brain

deUngria, Marissa; Rao, Raghavendra; Wobken, Jane D.; Luciana, Monica; Nelson, Charles A.; Georgieff, Michael K.

Perinatal iron deficiency decreases cytochrome c oxidase (CytOx) activity in selected regions of neonatal rat brain.

Intrauterine growth retardation and diabetes mellitus during human gestation result in significant losses of fetal and neonatal brain iron. Brain iron deficiency is associated with impaired cognitive processes including memory and attention. The regional distribution of iron staining and cytochrome c oxidase (CytOx) activity have not been mapped in the iron-sufficient or -deficient neonatal rat. CytOx is the iron-containing terminal enzyme in oxidative phosphorylation; its activity reflects neuronal metabolism. We hypothesized that neonatal brain iron deficiency differentially decreases iron and CytOx activity in brain regions, with more pronounced losses in structures involved in recognition memory. Pregnant Sprague Dawley rats were fed either an iron-deficient or -fortified diet from gestational d 1 until postnatal d 10. Iron staining and CytOx activity of 20 brain structures were mapped histochemically in 25 rats from each group. Brain iron staining was reduced from 75% to 100% and CytOx staining was decreased from 0% to 42% in the iron deficient group (p < 0.001). Areas with significantly reduced CytOx activity (p < 0.001) included all measured subareas of the hippocampus (CA1: 42%, CA3ab: 34%, CA3c: 33%, and dentate gyrus: 32%), the piriform cortex (17%), the medial dorsal thalamic nucleus (28%), and the cingulate cortex (41%). In contrast, the anterior thalamic nucleus, the lateral amygdaloid nucleus, and the medial habenula, areas not involved in higher cognitive functions, did not have significantly reduced CytOx activity (0%, 10%, and 16%, respectively). We conclude that perinatal iron deficiency differentially reduces neuronal metabolic activity, specifically targeting areas of the brain involved in memory processing.

MtDNA replicative potential remains constant during ageing: polymerase gamma activity does not correlate with age related cytochrome oxidase activity decline in platelets.

Progressive age-related oxidative phosphorylation (OxPhos) decline is well known in human tissues. Depletion of mitochondrial DNA (mtDNA) causes OxPhos defects in patients with myopathic syndromes and deficient mtDNA replication has been observed in cells cultured from patients with mitochondrial disease. Patients undergoing treatment for AIDS develop OxPhos defects via mtDNA depletion resulting from inhibition of mtDNA polymerase gamma (Polgamma) by 2'-deoxy 3'-azido thymidine. These findings by others give rise to a possible link between mtDNA replication and bioenergetic decline in disease and during ageing. We have designed an in vitro assay for Polgamma function in small tissue samples to explore this possible link. Platelet homogenate Polgamma showed an activity with a K m of 150 microM (dTTP), a V max of 11.8 pmol/min/mg, inhibited (41% inhibition; 50 microM) by ethidium bromide. Determination of several storage characteristics showed that platelets were a convenient source of Polgamma for assay. Polgamma activity in 45 subjects did not coincide with significant age-related decline (P<0.002; P) observed in cytochrome oxidase (CytOx) activity or with citrate synthase activity. Of the activities studied, the only significant age-wise variation was a 24% CytOx deficiency in elderly (>50; n = 19) compared to young (<51; n = 24) individuals (P<0.01; t). These results suggest a maintenance of total cellular mtDNA Polgamma processive levels during ageing, largely independent of total cellular bioenergetic status or mitochondrial number/density. The processive component of Polgamma is therefore unlikely to make a major contribution to age-related bioenergetic activity decline. This does not, however, preclude the possibility that transient periods of inhibition at crucial points of the cell cycle or development may augment existing intracellular deficiencies. The assay described here greatly facilitates study of Polgamma activity in patients with conditions involving mtDNA depletion or rearrangement.

Lipid peroxidation and changes in cytochrome c oxidase and xanthine oxidase activity in organophosphorus anticholinesterase induced myopathy

A possible role of radical oxygen species (ROS) initiated lipid peroxidation in diisopropylphosphorofluoridate (DFP)-induced muscle necrosis was investigated by quantifying muscle changes in F 2-isoprostanes, novel and extremely accurate markers of lipid peroxidation in vivo. A significant increase in F 2-isoprostanes of 56% was found in the diaphragm of rats 60 min after DFP-induced fasciculations. As possible source of ROS initiating lipid peroxidation, the cytocrome-c oxidase (Cyt-ox) and xanthine dehydrogenasexanthine oxidase (XD-XO) systems were investigated. Within 30 min of onset of fasciculations Cyt-ox activity was reduced by 50% from 0.526 to 0.263 μmol/mg prot/min and XO activity increased from 0.242 to 0.541 μmol/mg prot/min. Total XD-XO activity was unchanged, indicating a conversion from XD into XO. In rats pretreatment with the neuromuscular blocking agent d-tubocurarine, prevented DFP-induced fasciculations, increases in F 2-isoprostanes and changes in Cyt-ox or XD-XO. The decrease in Cyt-ox and increase in XO suggest that ROS are produced during DFP induced muscle fasciculations initiating lipid peroxidation and subsequent myopathy.

Effect of microgravity on the expression of mitochondrial enzymes in rat cardiac and skeletal muscles.

The purpose of this study was to examine the expression of nuclear and mitochondrial genes in cardiac and skeletal muscle (triceps brachii) in response to short-duration microgravity exposure. Six adult male rats were exposed to microgravity for 6 days and were compared with six ground-based control animals. We observed a significant 32% increase in heart malate dehydrogenase (MDH) enzyme activity, which was accompanied by a 62% elevation in heart MDH mRNA levels after microgravity exposure. Despite modest elevations in the mRNAs encoding subunits III, IV, and VIc as well as a 2.2-fold higher subunit IV protein content after exposure to microgravity, heart cytochrome c oxidase (CytOx) enzyme activity remained unchanged. In skeletal muscle, MDH expression was unaffected by microgravity, but CytOx activity was significantly reduced 41% by microgravity, whereas subunit III, IV, and VIc mRNA levels and subunit IV protein levels were unaltered. Thus tissue-specific (i.e., heart vs. skeletal muscle) differences exist in the regulation of nuclear-encoded mitochondrial proteins in response to microgravity. In addition, the expression of nuclear-encoded proteins such as CytOx subunit IV and expression of MDH are differentially regulated within a tissue. Our data also illustrate that the heart undergoes previously unidentified mitochondrial adaptations in response to short-term microgravity conditions more dramatic than those evident in skeletal muscle. Further studies evaluating the functional consequences of these adaptations in the heart, as well as those designed to measure protein turnover, are warranted in response to microgravity.

PERINATAL IRON DEFICIENCY DECREASES CYTOCHROME OXIDASE (CytOx) ACTIVITY IN SPECIFIC REGIONS OF NEONATAL RAT BRAIN † 1721

Brain iron deficiency bodes poorly for cognitive neurodevelopment and is present in newborns following gestations complicated by diabetes mellitus or intrauterine growth retardation. CytOx is an iron-containing enzyme involved in oxidative phosphorylation; its activity reflects neuronal metabolism. Regional CytOx activity has been mapped in the iron-sufficient adult rat brain but not in iron-sufficient or -deficient neonates. We hypothesized that neonatal brain iron deficiency alters regional CytOx activity. Pregnant Sprague-Dawley rats were fed iron-deficient or -sufficient chow from GD 1 until PND 7. CytOx activity in 14 brain nuclei (9 involved in memory pathways) was measured histochemically utilizing diaminobenzidine in frozen 15 μ coronal brain sections located approximately 2 mm posterior to the auditory meatus in 13 iron-deficient and 11 iron-sufficient pups. Quantitation of optical density (OD) was by Image-1. Brain iron concentration was 45% lower(p<0.001) with 13% less overall CytOx staining (p<0.001) in the iron-deficient group. CytOx activity loss in the iron- deficient group was not homogeneous; areas with reduced CytOx activity included the caudate/putamen(OD units: 94.8 ± 24.6 v 128.3 ± 33.1; p=0.02), the lateral amygdala (90.1 ± 17.3 v 119.3 ± 35.8; p=0.03) and the hippocampal CA1 region (151 ± 15 v 168 ± 15; p=0.05), while the cingulum, the medial and lateral habenula, and hippocampal areas CA3ab and CA3c of the hippocampus showed no loss. The piriform cortex, the lateral, ventral and dorsomedial thalamic nuclei, the periventricular arcuate hypothalamus and the dentate gyrus showed trends toward losses(p=0.06-0.19). The hippocampus is central to recognition memory circuits, the caudate-putamen and the piriform cortex to working/spatial memory circuits, and the amygdala and cingulum to emotional memory circuits. Perinatal iron deficiency has a non-homogeneous effect on neuronal metabolic activity, with certain nuclei involved in memory processing showing increased vulnerability. This factor may contribute to the poorer long-term developmental outcome observed among infants born following pregnancies complicated by gestational diabetes or intrauterine growth retardation. Supported by NICHD.

PERINATAL BRAIN IRON DEFICIENCY INCREASES THE VULNERABILITY OF RAT HIPPOCAMPUS TO HYPOXIC ISCHEMIC INSULT. † 1419

Postnatally acquired dietary iron deficiency has adverse long-term effects on neurodevelopment. Recently, our laboratory has identified gestational conditions (diabetes mellitus, intrauterine growth retardation) which result in a significant prevalence of perinatal brain iron deficiency. These groups of infants have poorer long-term developmental outcome, but the role of iron deficiency is unknown. Loss of cytochrome oxidase (CytOx) activity in the region of the hippocampus, an area central to cognitive function (recognition memory), has been shown to be an early and sensitive indicator of neuronal damage following perinatal hypoxic-ischemic encephalopathy (HIE) in the rat. We hypothesized that perinatal iron deficiency enhances the vulnerability of the hippocampus to HIE injury. Twenty 7-day-old, iron-deficient rat pups and 20 iron-sufficient controls underwent right carotid artery ligation and were exposed to 2.5 hours of hypoxia (8% FIO2). In this model (the Levine model), both hypoxia and ischemia (right side), but not hypoxia alone (left side), are needed to induce changes in neuronal CytOx activity. The left side is thus the internal control. The mean ± SD brain iron concentration of the iron-deficient group was 40% lower than the iron-sufficient group (28.0± 6.8 v 51.0 ± 7.8; p<0.001). The CytOx activity, visualized with diaminobenzedine, in four different areas of hippocampus 24 hours after hypoxic-ischemic damage was measured in optical density (OD) units (range: 1 black/254 white) by computer-assisted densitometry. There was right-sided CytOx activity loss in the dentate gyrus (102 ± 16 v 89 ± 17 OD units; p=0.009), CA3c (105 ± 16 v 93 ± 19 OD units; p=0.003) and CA1 (108 ± 27 v 98 ± 17 OD units; p=0.06) compared to the unligated left side in the iron-deficient group. Side-to-to-side differences were seen only in CA1 in the iron-sufficient group. Overall, the iron-deficient group had more subareas with right-sided loss than the iron-sufficient group (p=0.04), and the magnitude of right-left differences was greater in the iron-deficient group (p=0.05). Perinatal iron deficiency enhances the vulnerability of the neonatal brain to HIE and may contribute to long-term neurodevelopmental delays in at-risk newborns. Supported by NICHD.

Effects of Elevated Carbon Dioxide on Key Mitochondrial Respiratory Enzymes in `Hass' Avocado Fruit and Fruit Disks

Preclimacteric avocado [Persea americana (Mill.) cv. Hass] fruit or fruit disks as well as fruit harvested in either June (midseason) or August (late season) and partially ripened were kept in air (21% O2 + 78% N2), 20% CO2 + 17% O2 (63% N2), or 40% CO2 + 13% O2 (47% N2) at either 10 or 20 °C. Ethylene production by preclimacteric fruit was completely inhibited during CO2 exposure, whereas there was only partial inhibition of ethylene production when partially ripened fruit were exposed. Compared to the fruit stored in air, O2 uptake of fruit stored in 20% CO2 was decreased by 20%, whereas the fruit stored in 40% CO2 showed 25% more O2 uptake than air-stored fruit. Fruit subjected to a storage regime of 40% CO2 at 10 °C followed by 2 d in air had the best visual quality. In general, climacteric fruit treated with 20% CO2 at 10 °C showed increased pyruvate dehydrogenase (PDH) activity and decreased cytochrome oxidase (CytOx) activity. Fruit stored in 40% CO2 had reduced CytOx activity compared to air-stored fruit, and PDH activity was variable depending on the harvest season of the fruit. Our results show that the effect of elevated CO2 on a given enzyme depends on concentration of CO2, duration of exposure, physiological state of the fruit, and type of tissue exposed.

Effects of Endurance Training on Skeletal Muscle Oxidative Capacities with and without Selenium Supplementation

The purpose of this study was to examine the changes induced by endurance training, with or without selenium (Se) supplementation on: 1) mitochondrial activity of succinate dehydrogenase (SDH) and cytochrome c oxidase (Cyt Ox),2) the myosin heavy chain (MHC) expression in muscle fibers and 3) their association with aerobic performance. Twenty-four male students volunteered to participate in this double blind study: selenium (Sel, N = 12) vs placebo (Pla, N = 12). During a 10-wk endurance training program, the Sel group received a daily Se supplementation containing 180 micrograms of organic selenium (selenomethionine), while the Pla group received a placebo. Before (Pre) and after (Post) the program (3 sessions wk-1) an endurance exercise (Capmax) was performed in order to determine the aerobic endurance capacity assessed by the total oxygen uptake during the running test (VO2tot). All parameters of aerobic performance were increased in both groups, concomitantly to a rise in mitochondrial Cyt Ox activity. Two positive relationships were found: 1) between type I MHC and VO2tot increments (r = 0.65, P < 0.05), 2) between training volumes and VO2tot increments (r = 0.53, P < 0.05; N = 23). The training program produced an 8.2% significant increase in type I MHC (P < 0.05) while type II MHC decrease was not significant (-4.4%). Although they were almost non-existent before the program, muscle fibers which co-expressed type I and II MHC displayed a marked increase afterwards (4.9 +/- 5.7 vs 1.1 +/- 2.1%, P < 0.05). Muscle GSH-Px activity, at rest, did not respond to endurance training or Se supplementation. The results suggest that the neuromuscular system is still in an evolutive state after 10 weeks of endurance training, and that selenium supplementation has no effect on endurance training-induced adaptations.

Effects of hypothyroidism and aortic constriction on mitochondria during cardiac hypertrophy

We evaluated mitochondrial adaptations in the hearts of euthyroid and hypothyroid rats subject to aortic constriction for 2, 4, 7, 14, 21, and 28 d to induce a pressure-overload (PO), compared to sham-operated (SH) controls. PO animals attained higher arterial pressures than SH animals, by 55% in the euthyroid group, but only 14% in hypothyroid rats after 28 d. The left ventricle/body weight ratio was increased 44% by PO in the euthyroid group, and 26% in the hypothyroid group. PO attenuated the decline in cardiac growth in the hypothyroid group. Thus, hypothyroidism reduces the magnitude of the PO, but not the potential for hypertrophy in response to PO. Cytochrome c oxidase activity (CYTOX) was unchanged by PO in the euthyroid animals, indicating that the synthesis of mitochondria paralleled adaptive growth. However, CYTOX activity decreased up to 20% in the hypothyroid groups (P < 0.05) and was unaltered by PO. Thus, PO prevented the decline in growth, but not the decline in mitochondrial enzymes due to hypothyroidism. The lack of effect of PO on mitochondria was partly due to pretranslational changes since CYTOX subunit VIc mRNA was reduced by PO in the hypothyroid animals, but not in the euthyroid group. Levels of the chaperones HSP60 and GRP75, as well as HSP60 mRNA were unaffected by hypothyroidism, but paralleled adaptive growth induced by PO. Hypothyroidism changes the pattern of gene expression within the heart leading to altered mitochondrial composition. This cannot be compensated for by conditions of increased physiological demand.

Respiration of `Hass' Avocados in Response to Elevated CO2 Levels

Stress levels of carbon dioxide can be effective in the retardation of ripening and control of decay-causing pathogens and insect infestation of some horticultural perishables. Our objective has been to identify key mitochondrial enzymes and pathways that regulate the fruit's response to CO2 actions. Oxygen uptake of fruit stored in air + 20% CO2 (16.8% O2) was depressed compared to the airstored fruit, whereas the fruit stored in air + 40% CO2 (12.6% O2) had an elevated respiration rate. Climacteric fruit treated with 20% CO2 at 10C had increased pyruvate dehydrogenase (PDH) activity, decreased cytochrome oxidase (CytOx) activity, and double the alternative oxidase (AltOx) activity compared to air-stored fruit. Air + 40% CO2-stored fruit had reduced PDH and CytOx activities, and 50% more AltOx activity than the control fruit. Mitochondria were treated directly with the same CO2-enriched atmospheres to measure the catalytic effects of CO2. Total O2 uptake was decreased in both CO2 atmospheres and the cytochrome/alternative pathway ratio was greater than with mitochondria held in air. Nuclear magnetic resonance analysis of whole fruit confirmed that these CO2 atmospheres decrease the intracellular pH several 0.1 pH units with 2 h.

373 MITOCHONDRIAL RESPIRATlON OF `HASS' AVOCADOS IN RESPONSE TO ELEVATED CO, CONCENTRATIONS

Carbon dioxide-enriched atmospheres can be effective in the retardation of ripening and in the reduction of decay of horticultural commodities. However, concentrations in excess of the tolerance level may cause physiological damage. The goal of our research is to elucidate the specific regulatory mechanisms of CO2 actions. Cytochrome oxidase (CytOx) in vitro activity in preclimacteric avocado fruit stored in air or 40% CO2 + 12.6% O2 was evaluated at 20C. Activities were determined during treatment and also after a transfer to air. Fruit treated with 40% CO2 + 12.6% O2 had elevated CytOx in vitro activity when compared to air-stored fruit. Immunoblot analysis was performed to determine if the increase in CytOx activity could be due to an increase in enzyme concentration. The decline in respiration rate of CO,-treated fruit was most likely due to the decrease in intracellular pH and its effect on the activities of important respiratory enzymes, including CytOx. The regulatory mechanisms of other mitochondrial respiratory enzymes in `Hass' avocados exposed to elevated CO2 atmospheres are also under investigation.

Oxidative and glycolytic pathways in rat (newborn and adult) and turtle brain: role during anoxia

Using enzyme histochemistry and in vitro electrophysiological recordings in brain slices, we studied 1) the relative activity of cytochrome c oxidase (Cytox) and hexokinase (HK) and 2) cellular function by examining ionic homeostasis across cell membranes in the turtle and newborn (5 days old) and adult rat central nervous system. We found that Cytox was higher in the rostral than in the caudal brain regions of the adult rat and that the activity in the newborn is at least as high as in the adult rat. In contrast, adult turtles had very low Cytox activity throughout the central nervous system. Compared with that in the adult rat, HK activity in the newborn was generally lower in the rostral brain and cerebellum but similar or higher in the brain stem and spinal cord. In the turtle, HK activity was higher in the cerebellum, brain stem, and ventral horn of the spinal cord than in those in the rat. During anoxia, extracellular K+ increased by approximately 10-fold (from 3.2 to approximately 32 mM) in the adult brain stem but only by 2.6 mM in newborn rats. After glycolysis was blocked with iodoacetic acid (10-20 mM), extracellular K+ increased remarkably in both adult and newborn rats to approximately 35 mM. In contrast, the turtle brain tissue showed a slight and insignificant increase in extracellular K+ during complete anoxia or with iodoacetic acid; there was a modest increase in K+ when anoxia and iodoacetate were administered together. We conclude that 1) the newborn rat brain must rely either on higher glycolytic capacity or on a reduction of metabolic rate during O2 deprivation and 2) the turtle brain can subsist on nonglucose fuels or on fuels not requiring the citric acid cycle and the electron transfer chain.

Summertime and early autumn activity of some enzymes in the carbohydrate and fatty acid metabolism of the crucian carp

In June, July, and September the activities of five enzymes involved in the carbohydrate and fatty acid metabolism, namely phosphofructokinase (PFK), lactate dehydrogenase (LDH), citrate synthase (CS), cytochrome oxidase (cyt ox) and 3-hydroxy-acyl-CoA dehydrogenase (HAD), were measured in the heart, liver, red muscle, white muscle, and gill lamellae of crucian carp (Carassius carassius). LDH activity was measured in both reducing (LDHr) and oxidizing (LDHo) directions.The PFK activity in red and white muscle increased significantly between July and September indicating a preparation to winter anoxia by an increased glycylytic capacity in these organs. The HAD activity of the liver had increased significantly (by more than 50%) by September, also a preparation to winter anoxia as HAD is used in the reversed β-oxidation (chain elongation of fatty acids). The LDHr and cyt ox activities in the heart and white muscle were highest in July. This shows that both the anaerobic and aerobic capacities are elevated in mid-summer when water temperature is high and oxygen concentration of the water could fluctuate greatly. The LDHo and CS activities in gill lamellae were lowest in July. The results show that the metabolism of crucian carp is under seasonal influence and that a preparation to winter hypoxia/anoxia could be detected in September.