The exogenous application of plant hormones and their analogues has been exploited to improve crop performance in the field. Protodioscin is a saponin whose steroidal moiety has some similarities to plant steroidal hormones, brassinosteroids. To test the possibility that protodioscin acts as an agonist or antagonist of brassinosteroids or other plant growth regulators, we compared responses of the weed species Bidens pilosa L. to treatment with protodioscin, brassinosteroids, auxins (IAA) and abscisic acid (ABA). Seeds were germinated and grown in agar containing protodioscin, dioscin, brassinolides, IAA and ABA. Root apex respiratory activity was measured with an oxygen electrode. Malondialdehyde (MDA) and antioxidant enzymes activities were assessed. Protodioscin at 48-240 μm inhibited growth of B. pilosa seedlings. The steroidal hormone 24-epibrassinolide (0.1-5 μm) also inhibited growth of primary roots, but brassicasterol was inactive. IAA at higher concentrations (0.5-10.0 μm) strongly inhibited primary root length and fresh weight of stems. ABA inhibited all parameters of seedling growth and also seed germination. Respiratory activity of primary roots (KCN-sensitive and KCN-insensitive) was activated by protodioscin. IAA and ABA reduced KCN-insensitive respiration. The content of MDA in primary roots increased only after protodioscin treatment. All assayed compounds increased APx and POD activity, with 24-epibrassinolide being most active. The activity of CAT was stimulated by protodioscin and 24-epibrassinolide. The results revealed that protodioscin was toxic to B. pilosa through a mechanism not related to plant growth regulator signalling. Protodioscin caused a disturbance in mitochondrial respiratory activity, which could be related to overproduction of ROS and consequent cell membrane damage.

Estrogen deficiency accelerates the development of several disorders including visceral obesity and hepatic steatosis. The predisposing factors can be exacerbated by drugs that affect hepatic lipid metabolism. The aim of the present work was to determine if raloxifene, a selective estrogen receptor modulator (SERM) used extensively by postmenopausal women, affects hepatic fatty acid oxidation pathways. Fatty acids oxidation was measured in the livers, mitochondria and peroxisomes of ovariectomized (OVX) rats. Mitochondrial and peroxisomal β-oxidation was inhibited by raloxifene at a concentration range of 2.5–25 μM. In perfused livers, raloxifene reduced the ketogenesis from endogenous and exogenous fatty acids and increased the β-hydroxybutyrate/acetoacetate ratio. An increase in 14CO2 production without a parallel increase in the oxygen consumption indicated that raloxifene caused a diversion of NADH from the mitochondrial respiratory chain to another oxidative reaction. It was found that raloxifene has a strong ability to react with H2O2 in the presence of peroxidase. It is likely that the generation of phenoxyl radical derivatives of raloxifene in intact livers led to the co-oxidation of NADH and a shift of the cellular redox state to an oxidised condition. This change can perturb other important liver metabolic processes dependent on cellular NADH/NAD+ ratio.

This study investigated the allelopathic potential of both aqueous fraction of Bracharia ruziziensis L. straws and trans-aconitic acid, a component of aqueous fraction on growth and physiological processes of the weed Ipomoea triloba L. Both aqueous fraction and aconitic acid at 250-2000 ppm concentrations influenced the germination and growth of I. triloba and caused similar changes in the respiratory activity of primary roots. They reduced KCN-sensitive respiration and increased the KCN-insensitive respiration. The highest concentration of both aqueous fraction and trans-aconitic acid increased the malondialdehyde and conjugated diene content in the primary roots of seedlings. The oxygen consumption from citrate oxidation in mitochondria isolated from primary roots was not affected. Thus, the water soluble compounds of B. ruziziensis were phytotoxic to I. triloba, inducing perturbations in respiratory activity and lipid peroxidation. Although trans-aconitic acid exerted similar effects to the aqueous fraction, it is not the main compound responsible for the effects of aqueous fraction in I. triloba, because its content is very little in this fraction.

Ten years after the pioneer initiative of the Viçosa University, the bachelor degree in biochemistry can still be considered a novelty in Brazil. It certainly represents a challenge mainly because of the intrincate interplay of biochemistry with severalwell-established disciplines and carreers. The UEM-Department of Biochemistry decided to accept this challenge and is now offering its bachelor degree in 2011. Central to the main phylosophy underlying the proposed curriculum were the following points: (a) broad training in exact sciences, thus recognizing the quantitative nature of biochemistry; (b) broad offer of biochemical disciplines aiming to prepare the students for careers invarious service and industry sectors in addition to academic research; (c) full recognition of the marriage between biochemistry and molecular biology, as epitomized by the current names of the learned societies.The curriculum encompasses a wide range of biochemical disciplines from the traditional ones to technological branches. Experimental skillswill be developed inlaboratories of biomolecules, molecular biology, enzymology and metabolism. Receptiveness in the community was higlly promissing as can be judged by the prompt approval by the university and government authories as well as by the relatively elevated number of candidates in the entrance examinations.

The p‐coumaric acid, a phenolic acid, occurs in several plant species and, consequently, in many foods and beverages of vegetable origin. Its antioxidant activity is well documented, but there is also a single report about an inhibitory action on the monocarboxylate carrier, which operates in the plasma and mitochondrial membranes. The latter observation suggests that p‐coumaric acid could be able to inhibit gluconeogenesis and related parameters. The present investigation was planned to test this hypothesis in the isolated and hemoglobin‐free perfused rat liver. Transformation of lactate and alanine into glucose (gluconeogenesis) in the liver was inhibited by p‐coumaric acid (IC50 values of 92.5 and 75.6 μM, respectively). Transformation of fructose into glucose was inhibited to a considerably lower degree (maximally 28%). The oxygen uptake increase accompanying gluconeogenesis from lactate was also inhibited. Pyruvate carboxylation in isolated intact mitochondria was inhibited (IC50 = 160.1 μM); no such effect was observed in freeze–thawing disrupted mitochondria. Glucose 6‐phosphatase and fructose 1,6‐bisphosphatase were not inhibited. In isolated intact mitochondria, p‐coumaric acid inhibited respiration dependent on pyruvate oxidation but was ineffective on respiration driven by succinate and β‐hydroxybutyrate. It can be concluded that inhibition of pyruvate transport into the mitochondria is the most prominent primary effect of p‐coumaric acid and also the main cause for gluconeogenesis inhibition. The existence of additional actions of p‐coumaric acid, such as enzyme inhibitions and interference with regulatory mechanisms, cannot be excluded. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:18–26, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20114

It has been suggested that active forms of quercetin (o-semiquinones) are able to oxidize NADH in mammalian cells. The purpose of this study was to investigate this proposition by measuring the beta-hydroxybutyrate to acetoacetate ratio as an indicator of the mitochondrial NADH/NAD(+) redox ratio in the isolated perfused rat liver. The NADH to NAD(+) ratio was reduced by quercetin; half-maximal reduction occurred at a concentration of 32.6 MM. Additionally, quercetin (25 to 300 MM) stimulated the Krebs cycle ((CO2)-C-14 production) and inhibited oxygen uptake (50 to 300 mu M). Low quercetin concentrations (25 mu M) stimulated oxygen uptake. The results of the present work confirm the hypothesis that quercetin is able to participate in the oxidation of NADH in mammalian cells, shifting the cellular conditions to a more oxidized state (prooxidant activity). Stimulation of the Krebs cycle was probably caused by the increased NAD(+) availability whereas the decreased NADH availability and the inhibition of mitochondrial energy transduction could be the main causes for oxygen uptake inhibition.

The actions of zymosan on hepatic microcirculation and on the cell membrane permeability were investigated using the multiple-indicator dilution technique. The experimental system was the perfused rat liver. [3H]Water, [3H]sucrose and [14C]urea or [14C]bicarbonate were simultaneously injected into the portal vein. Mean transit times, distribution spaces, variances, linear superpositions and transfer coefficients across the plasma membrane were calculated. Zymosan had no net effect on the great vessels space but increased the extracellular sucrose space and decreased the aqueous cell space. Zymosan impaired the flow-limited distribution and increased the normalized variances of all tracers. The increase in the portal pressure caused by zymosan results most probably from a constriction just after or at the exit of the sinusoids. Impairment of the flow-limited distribution of tracers in the sinusoidal bed indicates that zymosan induces the formation of permeability barriers, which could make the access of the solutes to transporters or enzymes located on the outer surface of the plasma membrane difficult.

The present study evaluates the effects of methotrexate (MTX) and chloroquine (CQ), and of combined MTX + CQ treatment, on the inflammatory response and on plasma and liver phosphatase and transaminase activities, employing an adjuvant-induced arthritis model in rats. Arthritis was induced by the intradermal injection of a suspension of Mycobacterium tuberculosis in mineral oil into the plantar surface of the hind paws. Development of the inflammatory response was assessed over a 21-day period. Animal groups received either: (i) MTX, administered i.p., weekly, in 0.15, 1.5, 3, 6 or 12 mg/kg doses; (ii) CQ, given intragastrically, in daily 25 or 50 mg/kg doses; or (iii) MTX + CQ, administered in two combinations (MTX1.5 mg/kg + CQ(50 mg/kg), or MTX6 mg/kg + CQ(50 mg/kg)). At the end of the experimental period, the animals were anesthetized and killed, blood and liver samples were collected and prepared for measurement of acid and alkaline phosphatase (AP, ALP), and aspartate (AST) and alanine aminotransferase (ALT) activities. MTX at 6 and 12 mg/kg reduced the inflammatory response while CQ had no effect. MTX6 mg/kg + CQ(50 mg/kg) reduced the inflammatory response similar to MTX12 mg/kg, without affecting the bone marrow. Plasma AP and liver ALP activities were very elevated in the arthritic rats. While MTX treatment partially reduced both plasma AP and liver ALP activities at all doses used in the arthritic rats, CQ treatment reduced plasma AP, but increased liver AP activity. MTX + CQ treatment decreased plasma AP and liver ALP activities in the arthritic rats to control values. Plasma and liver AST activities were unaltered in the arthritic rats, and were unaffected by treatment. However, plasma and liver ALT activities were significantly reduced in the arthritic rats. While MTX or CQ treatment did not alter plasma transaminase activity in the arthritic rats, after MTX + CQ treatment, plasma ALT activity returned to normal values. In conclusion, the present data suggest that MTX + CQ treatment provides more effective anti-inflammatory protection against adjuvant-induced arthritis than does MTX alone, reverting the alterations in enzyme activities induced by this inflammatory disease in rats.

Kielmeyera coriacea Mart is a medicinal plant of the Clusiacea (Guttiferae) family used by the native population of Brazil in the treatment of several tropical diseases such as malaria, schistosomiasis, leishmaniasis, and fungal or bacterial infections. Kielmeyera coriacea is also effective as an antidepressant drug. Extracts of the plant are rich in xanthones. Compounds of this class have been reported to inhibit mitochondrial energy metabolism. For this reason the action of the Kielmeyera coriacea extract on hepatic energy metabolism was investigated in the present work, using isolated rat liver mitochondria and the perfused rat liver. In perfused livers the extract (20-80 microg/ml) caused stimulation of oxygen consumption, inhibition of gluconeogenesis and stimulation of glycogenolysis and glycolysis. In isolated mitochondria the Kielmeyera coriacea extract (5-20 microg/ml) stimulated state IV respiration, reduced the ADP/O ratio and decreased the respiratory coefficient. The activities of succinate-oxidase, NADH-oxidase, NADH dehydrogenase and succinate dehydrogenase were inhibited. The ATPase of intact mitochondria was stimulated and the ATPase of uncoupled mitochondria was inhibited. The results of this investigation suggest that the Kielmeyera coriacea extract impairs the hepatic energy metabolism by acting as mitochondrial uncoupler and inhibitor of enzymatic activities linked to the respiratory chain. The impairment of mitochondrial energy metabolism could lead to adverse metabolic effects by the use of the crude extract, but it could equally be the basis of its antiprotozoan and antifungal effects.

The action of NAD+ on hemodynamics and metabolism of the isolated perfused rat liver was investigated. Extracellular NAD+ (20–100 μM) stimulated glycogen breakdown (glucose release) and inhibited oxygen uptake. Lactate production was predominantly increased, and pyruvate production was predominantly inhibited. NAD+ also increased the portal perfusion pressure. All metabolic effects were strictly Ca2+-dependent. The effects were absent when Ca2+ was excluded, and reintroduction of the cation restored the effects. In preloaded livers, NAD+ accelerated 45Ca2+ efflux. The action of NAD+ was sensitive to three inhibitors of eicosanoid synthesis, suggesting that this action is mediated by these compounds, which are known to be produced and released by Kupffer and endothelial cells. It is impossible to infer from the available data if NAD+ exerts all these effects by itself or if they are caused by one or more of its extracellular hydrolysis products. Nicotinamide was ineffective and can be excluded, but especially cyclic ADP-ribose and ADP-ribose are possibilities that should be considered in future work.

There is substantial evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) affect cellular processes regulated by Ca(2+) ions, including the metabolic responses of the liver to Ca(2+)-dependent hormones. The aim of the present study was to determine whether the effects of naproxen are mediated by a direct action on cellular Ca(2+) fluxes. The effects of naproxen on 45Ca(2+) fluxes in mitochondria, microsomes and inside-out plasma membrane vesicles were examined. Naproxen strongly impaired the mitochondrial capacity to retain 45Ca(2+) and inhibited also ATP-dependent 45Ca(2+) uptake by microsomes. Naproxen did not modify 45Ca(2+) uptake by inside-out plasma membrane vesicles, but it inhibited the hexokinase/glucose-induced Ca(2+) efflux from preloaded vesicles. Additional assays performed in isolated mitochondria revealed that naproxen causes mitochondrial uncoupling and swelling in the presence of Ca(2+) ions. These effects were prevented by EGTA, ruthenium red and cyclosporin A, indicating that naproxen acts synergistically with Ca(2+) ions by promoting the mitochondrial permeability transition. The experimental results suggest that naproxen may impair the metabolic responses to Ca(2+)-dependent hormones acting by at least two mechanisms: (1) by interfering with the supply of external Ca(2+) through a direct action on the plasma membrane Ca(2+) influx, and (2) by affecting the refilling of the agonist-sensitive internal stores, including endoplasmic reticulum and mitochondria.

The effects of alpha-pinene, which is one of the major components of essential oils of several aromatic species, on energy metabolism of mitochondria isolated from maize (Zea mays L.) coleoptiles and primary roots were investigated. alpha-Pinene exerted similar effects on oxygen consumption irrespective of the source of mitochondria or of the substrate (L-malate, succinate or NADH). At concentrations lower than 250 muM, alpha-pinene stimulated respiration in state IV and inhibited respiration in state III. At higher concentrations the effect of alpha-pinene on state IV respiration was shifted toward inhibition. Complete suppression of respiratory control ratio was evident at alpha-pinene concentrations higher than 100 muM. When mitochondria were uncoupled with carbonyl cyanide 4-trifluoromethoxyphenyl-hydrazone (FCCP), alpha-pinene caused only inhibition of respiration. In the presence of alpha-pinene, the transmembrane potential was decreased as indicated by changes in the safranine binding by energized mitochondria. alpha-Pinene did not affect the activities of succinate dehydrogenase (EC 1.3.5.1) and L-malate dehydrogenase (L-malate:NAD(+) oxidoreductase; EC 1.1.1.37). The results indicate that alpha-pinene acts by at least two mechanisms: uncoupling of oxidative phosphorylation and inhibition of electron transfer. Confirming the impairment of mitochondrial energy metabolism, alpha-pinene strongly inhibited mitochondrial ATP production. It is apparent that the actions of alpha-pinene on isolated mitochondria are consequences of unspecific disturbances in the inner mitochondrial membrane. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

The effects of six allelochemicals from two different classes, phenolic acids and terpenes, were investigated on the respiratory activity of mitochondria isolated from soybean (Glycine max L.) hypocotyl axes. At 0.1 to 10.0 mM, the phenolic derivatives ferulic, coumaric and vanillic acids did not affect the mitochondrial respiration and related parameters (ADP/O ratio and respiratory control ratio) dependent on mixture of substrates composed of NADH, L-malate, succinate and L-glutamate. The effects and the potency of the monoterpenes were variable. alpha-Pinene stimulated respiration at low concentration (0.1 mM) but inhibited it at higher concentration both in the absence (basal respiration) and presence of ADP (coupled respiration), Limonene at 1.0 and 5.0 mM, inhibited the coupled respiration, but did not affect basal respiration. Camphor was less active and stimulated the basal respiration at 10.0 mM. It was concluded that the alpha-pinene and limonene may cause phytotoxicity to mitochondrial respiration. On the other hand, it is unlikely that the interference caused by ferulic, p-coumaric and vanillic acids on soybean growth and development is due to their effects on mitochondrial energy metabolism.

1. The influence of quercetin on glycogen catabolism and related parameters was investigated in the isolated perfused rat liver and subcellular systems. 2. Quercetin stimulated glycogenolysis (glucose release). This effect was already evident at a concentration of 50 microM maximal at 300 microM and declined at higher concentrations. Quercetin also stimulated oxygen consumption, with a similar concentration dependence. 3. Lactate production from endogenous glycogen (glycolysis) was diminished by quercetin without significant changes in pyruvate production. 4. Quercetin did not inhibit glucose transport into cells but decreased intracellular sequestration of [5-(3)H]glucose under conditions of net glucose release. 5. In isolated mitochondria, quercetin diminished the energy transduction efficiency. It also inhibited several enzymatic activities, e.g. the K(+)-ATPase/Na(+)-ATPase of plasma membrane vesicles and the glucose 6-phosphatase of isolated microsomes. 6. No significant changes of the cellular contents of AMP, ADP and ATP were found. The cellular content of glucose 6-phosphate, however, was increased (3.12-fold). 7. Some of the effects of quercetin (glycogenolysis stimulation) can be attributed to its action on mitochondrial energy metabolism, as, for example, uncoupling of oxidative phosphorylation. However, the multiplicity of the effects on several enzymatic systems certainly produces an intricate interplay that also generates complex and apparently contradictory effects.

According to previous reports, adjuvant-induced arthritic rats present reduced activities of the hepatic glucose 6-phosphatase. A kinetic study was done in order to characterize this phenomenon. Microsomes were isolated from livers of arthritic and control rats (Holtzman strain) and the glucose 6-phosphatase was measured at various temperatures (13-37 degrees C) and glucose 6-phosphate concentrations. Irrespective of the temperature, the enzyme from arthritic rats presented a reduction of both V(max) and K(M). Detergent treatment of liver microsomes from control rats increased the activity, but no increase was found when microsomes from arthritic rats were treated in the same way. The mannose 6-phosphatase activity of detergent-treated microsomes from arthritic rats was only 25% of the activity found with detergent-treated microsomes from control rats. Without detergent treatment, the mannose 6-phosphatase activities of both control and arthritic rats were minimal. The activation energy, derived from V(max), was not changed by arthritis. In vivo arthritic rats presented higher hepatic glucose 6-phosphate concentrations, a phenomenon that is consistent with a reduced activity of glucose 6-phosphatase. It was concluded that in arthritic rats, the hydrolase is probably reduced, without a similar change in the translocase activity.

1. The action of quercetin on glucose catabolism and production was investigated in the perfused rat liver. 2. Quercetin inhibited lactate production from glucose: 80% inhibition was found at a quercetin concentration of 100 micro M, and at higher concentrations inhibition was complete. 3. Pyruvate production from glucose presented a complex pattern, but stimulation was evident at 100 and 300 micro M quercetin. Oxygen uptake tended to be increased. 4. Glucose synthesis from lactate and pyruvate was inhibited. Inhibition was already evident at 50 micro M quercetin and almost complete at 300 micro M. Concomitantly, the increment in oxygen uptake caused by lactate plus pyruvate was stimulated by 50 micro M quercetin, but clearly inhibited by higher concentrations (100-500 micro M). 5. Glucose phosphorylation in the high-speed supernatant fractions of liver homogenates was inhibited by quercetin, but only at concentrations above 150 micro M. 6. It is concluded that quercetin can inhibit both glucose degradation and production and increase the cytosolic NAD(+)/NADH ratio. 7. These effects are likely to arise from many causes. Reduction of oxidative phosphorylation, inhibition of Na(+)-K(+)-ATPase, inhibition of glucokinase and inhibition of glucose 6-phosphatase could all contribute to the overall action of quercetin.