Identification of SH2B1ß as a focal adhesion protein that regulates focal adhesion size and number.

The adaptor protein SH2B1ß participates in regulation of the actin cytoskeleton during processes such as cell migration and differentiation. Here, we identify SH2B1ß as a new focal adhesion protein. We provide evidence that SH2B1ß is phosphorylated in response to phorbol 12-myristate 13-acetate (PMA)-induced protein kinase C (PKC) activation and show that PMA induces a rapid redistribution of SH2B1ß out of focal adhesions. We also show that growth hormone (GH) increases cycling of SH2B1ß into and out of focal adhesions. Ser161 and Ser165 in SH2B1ß fall within consensus PKC substrate motifs. Mutating these two serine residues into alanine residues abrogates PMA-induced redistribution of SH2B1ß out of focal adhesions, decreases SH2B1ß cycling into and out of focal adhesions in control and GH-stimulated cells, and increases the size of focal adhesions. By contrast, mutating Ser165 into a glutamate residue decreases the amount of SH2B1ß in focal adhesions and increases the number of focal adhesions per cell. These results suggest that activation of PKC regulates SH2B1ß focal adhesion localization through phosphorylation of Ser161 and/or Ser165. The finding that phosphorylation of SH2B1ß increases the number of focal adhesions suggests a mechanism for the stimulatory effect on cell motility of SH2B1ß.

Fluxes in central carbohydrate metabolism of source leaves in a fructan-storing C3 grass: rapid turnover and futile cycling of sucrose in continuous light under contrasted nitrogen nutrition status.

This work assessed the central carbohydrate metabolism of actively photosynthesizing leaf blades of a C3 grass (Lolium perenne L.). The study used dynamic (13)C labelling of plants growing in continuous light with contrasting supplies of nitrogen ('low N' and 'high N') and mathematical analysis of the tracer data with a four-pool compartmental model to estimate rates of: (i) sucrose synthesis from current assimilation; (ii) sucrose export/use; (iii) sucrose hydrolysis (to glucose and fructose) and resynthesis; and (iv) fructan synthesis and sucrose resynthesis from fructan metabolism. The contents of sucrose, fructan, glucose, and fructose were almost constant in both treatments. Labelling demonstrated that all carbohydrate pools were turned over. This indicated a system in metabolic steady state with equal rates of synthesis and degradation/consumption of the individual pools. Fructan content was enhanced by nitrogen deficiency (55 and 26% of dry mass at low and high N, respectively). Sucrose content was lower in nitrogen-deficient leaves (2.7 versus 6.7%). Glucose and fructose contents were always low (<1.5%). Interconversions between sucrose, glucose, and fructose were rapid (with half-lives of individual pools ranging between 0.3 and 0.8 h). Futile cycling of sucrose through sucrose hydrolysis (67 and 56% of sucrose at low and high N, respectively) and fructan metabolism (19 and 20%, respectively) was substantial but seemed to have no detrimental effect on the relative growth rate and carbon-use efficiency of these plants. The main effect of nitrogen deficiency on carbohydrate metabolism was to increase the half-life of the fructan pool from 27 to 62 h and to effectively double its size.

Crucial role of cytochrome P450 in hepatotoxicity induced by 2,3-dimethoxy-1,4-naphthoquinone in rats.

Quinone toxicity is induced by two principal mechanisms: arylation/alkylation and a redox cycle. We have previously shown that increases in intracellular levels of superoxide anion and cell death induced by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a redox cycling quinone, are enhanced by pretreatment of rat primary hepatocytes with cytochrome P450 inhibitors. This indicates a novel interaction of quinones with cytochrome P450, and is thus worthy of further investigation using an in vivo model. The aim of this study was to examine the effects of cytochrome P450 inhibitors on DMNQ-induced hepatotoxicity in rats. When DMNQ was administered intraperitoneally, the activities of serum alanine aminotransferase and aspartate aminotransferase were found to increase in a dose-dependent manner, indicating that hepatotoxicity was induced by treatment with DMNQ. Pretreatment with the cytochrome P450 inhibitors SKF-525A (SKF), cimetidine and ketoconazole potentiated the DMNQ-induced hepatotoxicity. The blood concentration of DMNQ was not affected by administration of SKF. Pretreatment with the antioxidant α-tocopherol almost completely attenuated the hepatotoxicity induced by DMNQ and by the combination of DMNQ with SKF. Levels of reduced glutathione in the liver were decreased and levels of oxidized glutathione were increased by treatment with DMNQ. These effects were potentiated by pretreatment with SKF. DMNQ-induced lipid peroxidation in the liver was also enhanced by pretreatment with SKF. Taken together, these results indicate that DMNQ-induced hepatotoxicity is augmented by inhibition of cytochrome P450 and that this augmentation is due to the enhancement of oxidative stress.

A metabolite of carcinogenic 2-acetylaminofluorene, 2-nitrosofluorene, induces redox cycling in mitochondria.

The present study was designed to confirm the recent proposal that 2-nitrosofluorene (2-NOF) as well as N-hydroxy-2-aminofluorene (N-OH-AF) induce a redox-cycle in rat liver mitochondria as part of the chronic toxic effects of the carcinogen 2-acetylaminofluorene (2-AAF). The formation of O2.- was demonstrated in submitochondrial particles by the formation of adrenochrome with NADH and succinate as respiratory substrates. 2-NOF was as effective as paraquat, a known redox-cycler, the lowest effective concentration being 0.4 nmol 2-NOF/mg protein. Experiments with isolated mitochondria showed that 2-NOF, in contrast to N-OH-AF, induces cyanide-resistant O2 consumption only in the presence of respiratory substrates, indicating that the reduction, but not the reoxidation, depends on a continuous flow of electrons through the respiratory chain of the mitochondrial membrane. Lipid peroxidation was estimated by the formation of thiobarbituric-acid-reactive substances. In comparison to the well-known prooxidant tert-butylhydroperoxide, 2-NOF was not significantly active. The results support the notion that 2-NOF induces oxidative stress by mitochondrial redox-cycling in vivo. Effects other than lipid peroxidation seem to be important for the chronic toxicity of 2-AAF.

Direct evidence for recycling of myeloperoxidase-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxy chromane, by ascorbate/dihydrolipoate in living HL-60 cells.

Myeloperoxidase (MPO)-catalyzed one-electron oxidation of endogenous phenolic constituents (e.g., antioxidants, hydroxylated metabolites) and exogenous compounds (e.g., drugs, environmental chemicals) generates free radical intermediates: phenoxyl radicals. Reduction of these intermediates by endogenous reductants, i.e. recycling, may enhance their antioxidant potential and/or prevent their potential cytotoxic and genotoxic effects. The goal of this work was to determine whether generation and recycling of MPO-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC), by physiologically relevant intracellular reductants such as ascorbate/lipoate could be demonstrated in intact MPO-rich human leukemia HL-60 cells. A model system was developed to show that MPO/H(2)O(2)-catalyzed PMC phenoxyl radicals (PMC*) could be recycled by ascorbate or ascorbate/dihydrolipoic acid (DHLA) to regenerate the parent compound. Absorbance measurements demonstrated that ascorbate prevents net oxidation of PMC by recycling the phenoxyl radical back to the parent compound. The presence of DHLA in the reaction mixture containing ascorbate extended the recycling reaction through regeneration of ascorbate. DHLA alone was unable to prevent PMC oxidation. These conclusions were confirmed by direct detection of PMC* and ascorbate radicals formed during the time course of the reactions by EPR spectroscopy. Based on results in the model system, PMC* and ascorbate radicals were identified by EPR spectroscopy in ascorbate-loaded HL-60 cells after addition of H(2)O(2) and the inhibitor of catalase, 3-aminotriazole (3-AT). The time course of PMC* and ascorbate radicals was found to follow the same reaction sequence as during their recycling in the model system. Recycling of PMC by ascorbate was also confirmed by HPLC assays in HL-60 cells. Pre-loading of HL-60 cells with lipoic acid regenerated ascorbate and thus increased the efficiency of ascorbate in recycling PMC*. Lipoic acid had no effect on PMC oxidation in the absence of ascorbate. Thus PMC phenoxyl radical does not directly oxidize thiols but can be recycled by dihydrolipoate in the presence of ascorbate. The role of phenoxyl radical recycling in maintaining antioxidant defense and protecting against cytotoxic and genotoxic phenolics is discussed.

Substrate cycling between pyruvate and oxaloacetate in awake normal and 3,3'-5-triiodo-L-thyronine-treated rats.

Substrate cycling between pyruvate and oxaloacetate was assessed in awake 24-h fasted normal and triiodothyronine (T3)-treated rats. After a 20- or 60-min infusion of [3-13C]alanine (99% enriched, 12 mg/min) the 13C enrichments of liver glucose and alanine carbons were analyzed by 13C and 1H nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. Substrate cycling from phosphoenolpyruvate to pyruvate [via pyruvate kinase (PK)] and from oxaloacetate to pyruvate [via malic enzyme (ME)] relative to the pyruvate carboxylase (PC) flux [i.e., (PK+ME)/PC] was assessed by the ratio of the 13C enrichment of C-2 alanine relative to that in C-5 glucose. In the normal rats (PK+ME)/PC was 0.26 +/- 0.07 (n = 7, t = 20 min) and 0.37 +/- 0.08 (n = 4, t = 60 min). In the T3-treated rats the (PK+ME)/PC increased four- to fivefold to 1.03 +/- 0.19 (n = 8, t = 20 min) and to 1.83 +/- 0.19 (n = 3, t = 60 min) (P < 0.05 vs. normal rats). The liver enzyme activity of PK did not change with T3 treatment (normal 14.22 +/- 5.25 U/g liver vs. T3 treated 13.40 +/- 1.10 U/g liver), whereas both the enzyme activity ratio of PK (normal 0.47 +/- 0.15 vs. T3 treated 0.77 +/- 0.03, P < 0.05) and the activity of ME (normal 0.89 +/- 0.30 U/g liver vs. T3 treated 4.25 +/- 0.60 U/g liver, P < 0.05) increased with T3 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of steroids on energy expenditure and substrate oxidation in women with Crohn's disease.

OBJECTIVE: Patients with Crohn's disease (CD) have increased energy expenditure and fat oxidation. Steroids, commonly used to treat flare-up of CD, induce weight gain. This study was designed to evaluate the effects of prednisone and budesonide on energy expenditure and substrate oxidation in patients with CD. METHODS: Twenty-nine women with CD and 10 healthy controls were studied. Ten patients received prednisone (0.75-1.0 mg/kg/day), nine received budesonide (9 mg/ day), and 10 did not receive steroids. Resting energy expenditure and substrate oxidation were measured by indirect calorimetry in a fasting state and after a standard diet. RESULTS: In the fasting state, resting energy expenditure was higher in patients without steroids than in the controls. Lipid oxidation was lower (p < 0.01) in patients with prednisone (0.46 +/- 0.39 mg/kg/min) than in patients with budesonide (0.97 +/- 0.28 mg/kg/min) and without steroids (1.06 +/- 0.32 mg/kg/min), but was similar with control subjects (0.47 +/- 0.20 mg/kg/min). Postprandially, lipid oxidation was lower (p < 0.01) in patients with prednisone (0.32 +/- 0.23 mg/kg/min) than in patients with budesonide (0.75 +/- 0.20 mg/kg/min), without steroids (0.82 +/- 0.23 mg/kg/min), and controls (0.58 +/- 0.15 mg/kg/min). Protein oxidation was significantly higher in patients with prednisone than in the other subjects. CONCLUSIONS: In women with CD, prednisone decreases lipid oxidation and increases protein oxidation. These effects are not observed with budesonide and may contribute to the weight gain and side effects commonly observed with prednisone. A low-fat/high-protein diet could be proposed during a course of prednisone.

Modifications of cardiac contractility by redox cycling alkylating and mixed redox cycling/alkylating quinones.

The effects of redox cycling, alkylating and mixed redox cycling/alkylating benzo- and naphthoquinones were examined in electrically driven guinea pig left atria. Cardiac microsomal and mitochondrial NAD(P)H-dependent metabolism of the quinones and consequent generation of superoxide anion (O2.-) were also measured. Mixed redox cycling/alkylating 2-methyl-1,4-naphthoquinone, redox cycling 2,3-dimethoxy-1,4-naphthoquinone and alkylating p-benzoquinone determined concentration-dependent positive inotropic responses, whereas redox cycling 2,3,5,6-tetramethyl-p-benzoquinone had no effect. The positive inotropic effect of 2,3-dimethoxy-1,4-naphthoquinone was completely catecholamine-mediated, that of 2-methyl-1,4-naphthoquinone was approximately 70% adrenergic and 30% direct. p-Benzoquinone acted directly on heart muscle. In time, quinones with alkylating properties caused increases in the resting force of atria, whereas redox cycling quinones did not produce toxic effects. Mitochondrial NADH-oxidoreductase accounted for 90 to 95% of the metabolism of all quinones, whereas the contribution of the microsomal pathway was negligible. Considerable amounts of O2.- were produced by mitochondrial biotransformation of 2-methyl-1,4-naphthoquinone and 2,3-dimethoxy-1,4-naphthoquinone but not of 2,3,5,6-tetramethyl-p-benzoquinone and p-benzoquinone, suggesting a kind of relation between O2.- generation and the release of catecholamines.