Search

Oxidative stress and inflammation are the key players in neuroinflammation, in which microglia dysfunction plays a central role. Previous studies suggest that argan oil attenuates oxidative stress, inflammation, and peroxisome dysfunction in mouse brains. In this study, we explored the effects of two major argan oil (AO) phytosterols, Schottenol (Schot) and Spinasterol (Spina), on oxidative stress, inflammation, and peroxisomal dysfunction in two murine microglial BV-2 cell lines, wild-ype (Wt) and Acyl-CoA oxidase 1 (Acox1)-deficient cells challenged with LPS treatment. Herein, we used an MTT test to reveal no cytotoxicity for both phytosterols with concentrations up to 5 µM. In the LPS-activated microglial cells, cotreatment with each of these phytosterols caused a significant decrease in intracellular ROS production and the NO level released in the culture medium. Additionally, Schot and Spina were able to attenuate the LPS-dependent strong induction of Il-1β and Tnf-α mRNA levels, as well as the iNos gene and protein expression in both Wt and Acox1−/− microglial cells. On the other hand, LPS treatment impacted both the peroxisomal antioxidant capacity and the fatty acid oxidation pathway. However, both Schot and Spina treatments enhanced ACOX1 activity in the Wt BV-2 cells and normalized the catalase activity in both Wt and Acox1−/− microglial cells. These data suggest that Schot and Spina can protect cells from oxidative stress and inflammation and their harmful consequences for peroxisomal functions and the homeostasis of microglial cells. Collectively, our work provides a compelling argument for the protective mechanisms of two major argan oil phytosterols against LPS-induced brain neuroinflammation.

Resveratrol is one of the best known polyphenol. While its effect on endothelial blood vessel cells, cancer cells, inflammatory processes and neurodegenerative events is well documented, only little is established on the metabolic implication of this phytophenol, particularly on skeletal muscle cells. Here, we report the effect of resveratrol on mouse skeletal muscle cells (C2C12) by measurement of cell proliferation, expression of metabolic genes and their transcription factor dependency, and modulation of non-coding microRNA expression. Resveratrol slightly decreases cell proliferation, while it up regulates PGC-1α a PPAR transcription factor co-activator involved in the control of metabolic genes. It also modulates microRNAs which are expressed in skeletal muscle cells and are involved in differentiation or contraction-relaxation. Results put forward new regulatory properties of resveratrol on skeletal muscles and highlight new potential applications of this molecule in muscle performances.

The mitochondrial-located enzyme Carnitine palmitoyltransferase [...]

The brain, which is a cholesterol-rich organ, can be subject to oxidative stress in a variety of pathophysiological conditions, age-related diseases and some rare pathologies. This can lead to the formation of 7-ketocholesterol (7KC), a toxic derivative of cholesterol mainly produced by auto-oxidation. So, preventing the neuronal toxicity of 7KC is an important issue to avoid brain damage. As there are numerous data in favor of the prevention of neurodegeneration by the Mediterranean diet, this study aimed to evaluate the potential of a series of polyphenols (resveratrol, RSV, quercetin, QCT, and apigenin, API) as well as &omega, 3 and &omega, 9 unsaturated fatty acids (&alpha, linolenic acid, ALA, eicosapentaenoic acid, EPA, docosahexaenoic acid, DHA, and oleic acid, OA) widely present in this diet, to prevent 7KC (50 &micro, M)-induced dysfunction of N2a neuronal cells. When polyphenols and fatty acids were used at non-toxic concentrations (polyphenols: &le, 6.25 &micro, M, fatty acids: &le, 25 &micro, M) as defined by the fluorescein diacetate assay, they greatly reduce 7KC-induced toxicity. The cytoprotective effects observed with polyphenols and fatty acids were comparable to those of &alpha, tocopherol (400 &micro, M) used as a reference. These polyphenols and fatty acids attenuate the overproduction of reactive oxygen species and the 7KC-induced drop in mitochondrial transmembrane potential (&Delta, &Psi, m) measured by flow cytometry after dihydroethidium and DiOC6(3) staining, respectively. Moreover, the studied polyphenols and fatty acids reduced plasma membrane permeability considered as a criterion for cell death measured by flow cytometry after propidium iodide staining. Our data show that polyphenols (RSV, QCT and API) as well as &omega, 9 unsaturated fatty acids (ALA, EPA, DHA and OA) are potent cytoprotective agents against 7KC-induced neurotoxicity in N2a cells. Their cytoprotective effects could partly explain the benefits of the Mediterranean diet on human health, particularly in the prevention of neurodegenerative diseases.

To clarify appropriateness of current claims for health and wellness virtues of argan oil, studies were conducted in inflammatory states. LPS induces inflammation with reduction of PGC1-αsignaling and energy metabolism. Argan oil protected the liver against LPS toxicity and interestingly enough preservation of peroxisomal acyl-CoA oxidase type 1 (ACOX1) activity against depression by LPS. This model of LPS-driven toxicity circumvented by argan oil along with a key anti-inflammatory role attributed to ACOX1 has been here transposed to model aging. This view is consistent with known physiological role of ACOX1 in yielding precursors of specialized proresolving mediators (SPM) and with characteristics of aging and related disorders including reduced PGC1-αfunction and improvement by strategies rising ACOX1 (via hormonal gut FGF19 and nordihydroguaiaretic acid in metabolic syndrome and diabetes conditions) and SPM (neurodegenerative disorders, atherosclerosis, and stroke). Delay of aging to resolve inflammation results from altered production of SPM, SPM improving most aging disorders. The strategic metabolic place of ACOX1, upstream of SPM biosynthesis, along with ability of ACOX1 preservation/induction and SPM to improve aging-related disorders and known association of aging with drop in ACOX1 and SPM, all converge to conclude that ACOX1 represents a previously unsuspected and currently emerging antiaging protein.

International audience; Phytophenols are considered to have beneficial effects towards human physiology. They are food microcomponents with potent chemopreventive properties towards the most three frequent contemporary human diseases, e.g., cardiovascular alterations, cancer and neurodegenerative pathologies. Related to this, the plasmatic form and plasmatic level of plant polyphenols in the body circulation are crucial for their efficiency. Thus, determinations of the binding process of resveratrol and of common flavonoids produced by major edible plants, berries and fruits to plasma proteins are essential. The interactions between resveratrol and albumin, a major plasma protein, were compared with those already published, involving curcumin, genistein, quercetin and other well-known food-containing polyphenols. The approaches used are usually intrinsic fluorescence intensity changes, quenching of protein intrinsic fluorescence and infrared spectroscopy. It appears that: (1) all of the studied polyphenols interact with albumin; (2) while most of the studied polyphenols interact at one albumin binding site, there are two different types of resveratrol binding sites for bovine serum albumin, one with the highest affinity (apparent KD of 4 µM) with a stoichiometry of one per monomer and a second with a lower affinity (apparent KD of 20 µM) with also a stoichiometry of one per monomer; (3) at least one binding site is in the vicinity of one tryptophanyl residue of bovine serum albumin; and (4) resveratrol binding to bovine serum albumin produces a very small structural conformation change of the polypeptide chain. These results support a role played by polyphenols-albumin interactions in the plasma for the bio-activities of these food microcomponents in the body.

Further advances in understanding the mechanism of action of resveratrol and its application require new analogs to identify the structural determinants for the cell proliferation inhibition potency. Therefore, we synthesized new trans-resveratrol derivatives by using the Wittig and Heck methods, thus modifying the hydroxylation and methoxylation patterns of the parent molecule. Moreover, we also synthesized new ferrocenylstilbene analogs by using an original protective group in the Wittig procedure. By performing cell proliferation assays we observed that the resveratrol derivatives show inhibition on the human colorectal tumor SW480 cell line. On the other hand, cell viability/cytotoxicity assays showed a weaker effects on the human hepatoblastoma HepG2 cell line. Importantly, the lack of effect on non-tumor cells (IEC18 intestinal epithelium cells) demonstrates the selectivity of these molecules for cancer cells. Here, we show that the numbers and positions of hydroxy and methoxy groups are crucial for the inhibition efficacy. In addition, the presence of at least one phenolic group is essential for the antitumoral activity. Moreover, in the series of ferrocenylstilbene analogs, the presence of a hidden phenolic function allows for a better solubilization in the cellular environment and significantly increases the antitumoral activity.

Since the first report by Rhodin in 1954, our knowledge on mammalian microbodies/peroxisomes has known several periods. An initial two decades period (1954–1973) has contributed to the biochemical individualisation of peroxisomes as a new class of subcellular organelles (de Duve, 1965). The corresponding research period failed to define a clear role of mammalian peroxisomes in vital functions and intermediary metabolism, explaining why feeling that peroxisomes might be in the human cell oddities has prevailed during several decades. The period standing from 1973 to nowadays has progressively removed this cell oddity view of peroxisomes by highlighting vital function and metabolic role of peroxisomes in health and disease along with genetic and metabolic regulation of peroxisomal protein content, organelle envelope formation and protein signal targeting mechanisms. Research on peroxisomes and their response to various drugs and metabolites, dietary and physiological conditions has also played a key role in the discovery of peroxisome proliferator activated receptors (PPARs) belonging to the nuclear hormone receptor superfamily and for which impact in science and medicine goes now by far beyond that of the peroxisomes.

Besides synthesizing nutritive substances (proteins, fats and carbohydrates) for energy and growth, plants produce numerous non-energetic so-called secondary metabolites (mainly polyphenols) that allow them to protect themselves against infections and other types of hostile environments. Interestingly, these polyphenols often provide cells with valuable bioactive properties for the maintenance of their functions and homeostasis (signaling, gene regulation, protection against acquired or infectious diseases, etc.) both in humans and animals. Namely, from a nutritional point of view, and based on epidemiological data, it is now well accepted that the regular consumption of green vegetables, fruits and fibers has protective effects against the onset of cancer as well as of inflammatory, neurodegenerative, metabolic and cardiovascular diseases, and consequently increases the overall longevity. In particular, grapevine plants produce large amounts of a wide variety of polyphenols. The most prominent of those-resveratrol-has been shown to impair or delay cardiovascular alterations, cancer, inflammation, aging, etc. Until recently, the molecular bases of the pleiotropic effects of resveratrol remained largely unclear despite numerous studies on a variety of signaling pathways and the transcriptional networks that they control. However, it has been recently proposed that the protective properties of resveratrol may arise from its modulation of small non-coding regulatory RNAs, namely microRNAs. The aim of this review is to present up-to-date data on the control of microRNA expression by dietary phytophenols in different types of human cells, and their impact on cell differentiation, cancer development and the regulation of the inflammatory response.

International audience; Resveratrol (3,4,5 trihydroxy-trans-stilbene) is one of the best known phytophenols with pleiotropic properties. It is a phytoalexin produced by vine and it leads to the stimulation of natural plant defenses but also exhibits many beneficial effects in animals and humans by acting on a wide range of organs and tissues. These include the prevention of cardiovascular diseases, anti-cancer potential, neuroprotective effects, homeostasia maintenance, aging delay and a decrease in inflammation. Age-related macular degeneration (AMD) is one of the main causes of deterioration of vision in adults in developed countries This review deals with resveratrol and ophthalmology by focusing on the antioxidant, anti-inflammatory, and anti-angiogenic effects of this molecule. The literature reports that resveratrol is able to act on various cell types of the eye by increasing the level of natural antioxidant enzymatic and molecular defenses. Resveratrol anti-inflammatory effects are due to its capacity to limit the expression of pro-inflammatory factors, such as interleukins and prostaglandins, and also to decrease the chemo-attraction and recruitment of immune cells to the inflammatory site. In addition to this, resveratrol was shown to possess anti-VEGF effects and to inhibit the proliferation and migration of vascular endothelial cells. Resveratrol has the potential to be used in a range of human ocular diseases and conditions, based on animal models and in vitro experiments.

In mammals, the liver is the major organ of fatty acid catabolism. This pathway is involved in both mitochondria and peroxisome. While mitochondria breaks down fatty acids with short, medium and long carbon chains, peroxisomes are involved in the catabolism of very long and branched chain fatty acids, which are degraded by three enzymes: acyl-CoA oxidase, multifunctional enzyme and thiolase enzyme. The active pathway results mainly from a tight transcriptional control of these gene-encoding enzymes. Two major nuclear receptors that are highly expressed in this organ are involved in this control, e.g. PPARα (peroxisome proliferator-activated receptor, α isoform) and HNF4α (hepatic nuclear factor 4, α isotype). Both are key regulators of liver lipid metabolism. While numerous papers have reported on the role of PPARα in liver lipid homeostasis, less is known on the implication of HNF4α in this metabolism. Moreover, very few studies have taken an interest in the important question of the implication of these two receptors and most particularly their crosstalk. This review therefore presents the current knowledge on the PPARα/HNF4α interplay in diversified DNA responsive elements and its relevance in the regulation fatty acid catabolism. It presents a review of the properties of the nuclear receptors PPARα and HNF4α, then the genes regulated by HNF4α and PPARα, particularly the peroxisomal enzyme target genes. To conclude, the consequences of the regulation of these genes in the liver by PPARα and HNF4α will be analyzed. The current data indicate the requirement of PPARα and HNF4α for regulation in the liver of peroxisomal and mitochondrial fatty acid β-oxidation, cholesterol and bile acid metabolism, lipoprotein metabolism and consequently the prevention of liver steatosis. However, several questions remain unsolved. To show the interplay of PPARα and HNF4α in the regulation of liver fatty acid metabolism, different strategies are proposed.

Numerous studies have reported interesting properties of trans-resveratrol, a phytoalexin, as a preventive agent of several important pathologies: vascular diseases, cancers, viral infections, and neurodegenerative processes. These beneficial effects of resveratrol have been supported by observations at the cellular and molecular levels in both cellular and in vivo models, but the cellular fate of resveratrol remains unclear. We suggest here that resveratrol uptake, metabolism, and stability of the parent molecule could influence the biological effects of resveratrol. It appears that resveratrol stability involves redox reactions and biotransformation that influence its antioxidant properties. Resveratrol's pharmacokinetics and metabolism represent other important issues, notably, the putative effects of its metabolites on pathology models. For example, some metabolites, mainly sulfate-conjugated resveratrol, show biological effects in cellular models. The modifications of resveratrol stability, chemical structure, and metabolism could change its cellular and molecular targets and could be crucial for improving or decreasing its chemopreventive properties.

10 pages; International audience; The peroxisomal 3-ketoacyl-CoA thiolase B (Thb) gene was previously identified as a direct target gene of PPARalpha, a nuclear hormone receptor activated by hypolipidemic fibrate drugs. To better understand the role of ThB in hepatic lipid metabolism in mice, Sv129 wild-type and Thb null mice were fed or not the selective PPARalpha agonist Wy14,643 (Wy). Here, it is shown that in contrast to some other mouse models deficient for peroxisomal enzymes, the hepatic PPARalpha signaling cascade in Thb null mice was normal under regular conditions. It is of interest that the hypotriglyceridemic action of Wy was reduced in Thb null mice underlining the conclusion that neither thiolase A nor SCPx/SCP2 thiolase can fully substitute for ThB in vivo. Moreover, a significant increased in the expression of lipogenic genes such as Stearoyl CoA Desaturase-1 (SCD1) was observed in Thb null mice fed Wy. Elevation of Scd1 mRNA and protein levels led to higher SCD1 activity, through a molecular mechanism that is probably SREBP1 independent. In agreement with higher SCD1, enrichment of liver mono-unsaturated fatty acids of the n-7 and n-9 series was found in Thb null mice fed Wy. Overall, we show that the reduced peroxisomal beta-oxidation of fat observed in Thb null mice fed Wy is associated with enhanced hepatic lipogenesis, through the combined elevation of microsomal SCD1 protein and activity. Ultimately, not only the amount but also the quality of the hepatic fatty acid pool is modulated upon the deletion of Thb.

International audience; 2,4-Dichlorophenoxyacetic acid (2,4D) is one of the widely used herbicide of the phenoxy family with possible startling number of adverse effects on species other than the weeds which is designed to kill. The effects of 2,4D were investigated in jerboa (Jaculus orientalis), a wild animal of subdesert highlands. The jerboas have been daily treated intraperitonally with 2,4D 3 mg/kg body weight for 4 weeks. Plasmatic markers, and antioxidants defences systems were assessed and histological alterations were evaluated. The in vivo and in vitro effects of 2,4D on the mitochondrial D-3-hydroxybutyrate dehydrogenase (BDH) were also determined. Our results showed a strong decrease of triglycerides level and HDL cholesterol and an increase in GOT level and LDL cholesterol. The microscopic evaluation showed that 2,4D induced necrosis of seminiferous tubules cells in testis, hyperplasia of hepatocytes in liver and presence of multinucleated giant cells in brain. The results show also an inhibitory effect on BDH in terms of activity and kinetic parameters. All of these results show that 2,4D induces toxicity which affects energy metabolism, morphological perturbation and oxidative stress.

The individual cytotoxicity of cadmium chloride, iron sulphate and chromium nitrate has been investigated by using the freshwater ciliate Tetrahymena pyriformis. The metabolic enzymes and antioxidant defense biomarkers were assessed. The results obtained reveal that their metal salts have perturbed the physiology and morphology of T. pyriformis. Also, the biomarkers assessed were sensitive to the presence of metal salts and this sensitivity was metal salt and dose dependant. To estimate the impact of their metal salts on mitochondria, we studied their effects in vivo and in vitro on the d -β-hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30) inner mitochondrial membrane enzyme. The results showed a high inhibition of BDH in terms of activity, protein expression and kinetic parameters.

In patients with sepsis, liver metabolism and its capacity to provide other organs with energetic substrates are impaired. This and many other pathophysiological changes seen in human patients are reproduced in mice injected with purified endotoxin (lipopolysaccharide, LPS). In the present study, down-regulation of genes involved in hepatic fatty acid oxidation (FAOx) and gluconeogenesis in mice exposed to LPS was challenged by nutritional intervention with Argan oil. Mice given a standard chow supplemented or not with either 6% (w/w) Argan oil (AO) or 6% (w/w) olive oil (OO) prior to exposure to LPS were explored for liver gene expressions assessed by mRNA transcript levels and/or enzyme activities. AO (or OO) food supplementation reveals that, in LPS-treated mice, hepatic expression of genes involved in FAOx and gluconeogenesis was preserved. This preventive protection might be related to the recovery of the gene expressions of nuclear receptors peroxisome proliferator-activated receptor α (PPARα) and estrogen related receptor α (ERRα) and their coactivator peroxisome proliferator-activated receptor gamma coactivator-1α, (PGC-1α). These preventive mechanisms conveyed by AO against LPS-induced metabolic dysregulation might add new therapeutic potentialities in the management of human sepsis., Highlights • Argan oil prevents LPS-treated mice from liver dysregulation of FAOx and gluconeogenesis. • Argan oil improves hepatic expression of PPARα and ERRα, and their coactivators PGC-1α and Lipin-1. • New preventive mechanisms conveyed by Argan oil against LPS-induced metabolic dysregulation.

Mitochondrial membrane-bound and phospholipid-dependent D-beta-hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30), a ketone body converting enzyme in mitochondria, has been studied in two populations of mitochondria (heavy and light) of jerboa (Jaculus orientalis) liver. The results reveal significant differences between the BDH of the two mitochondrial populations in terms of protein expression, kinetic parameters and physico-chemical properties. These results suggest that the beta-hydroxybutyrate dehydrogenases from heavy and light mitochondria are isoform variants. These differences in BDH distribution could be the consequence of cell changes in the lipid composition of the inner mitochondrial membrane of heavy and light mitochondria. These changes could modify both BDH insertion and BDH lipid-dependent catalytic properties.

The peroxisomal beta-oxidation system consists of four steps catalysed by three enzymes: acyl-CoA oxidase, 3-hydroxyacyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (multifunctional enzyme) and thiolase. In humans, thiolase activity is encoded by one gene, whereas in rodents, three enzymes encoded by three distinct genes (i.e. thiolase A, thiolase B and SCP2/thiolase) catalyse the thiolase activity. So far, acyl-CoA oxidase- and multifunctional enzyme-deficient patients have been identified and knock-out mice for these genes have been produced. Conversely, no isolated thiolase-deficient patient has been found, and no thiolase (A or B)-deficient mice have been generated. Hence, to better understand the cause of isolated human thiolase deficiency, we disrupted the catalytic site of the mouse thiolase B by homologous recombination in order to analyse the phenotype of these thiolase B-deficient mice. Mice, made homozygous for the mutation, lack expression of thiolase B mRNA and are viable, fertile and healthy at birth. They exhibit no detectable phenotype defects and no compensation, rather a slight decrease in other peroxisomal thiolase (thiolase A and SCPx) mRNAs, was found.

Resveratrol produced by several plants, berries and fruits, including grapes, is one of the best known natural food microcomponents with potent chemopreventive properties towards the most severe contemporary human diseases: cardiovascular sickness, cancer and neurodegenerative pathologies. Demonstration of its mechanism of action also implies the elucidation of the steps of bioavailability and bioabsorption in cells and tissues. In order to estimate the relationships between the amounts of resveratrol taken up by food or drink intake, and the several possible benefits illustrated from in vitro/in vivo experiments and from epidemiological studies, it is essential to demonstrate step by step the route of resveratrol from plasma to the cell active site. In plasma, resveratrol was shown to interact with lipoproteins. This commentary also contains previously unpublished results about interactions between resveratrol and albumin and the enhancement of this binding in presence of fatty acids. We have previously described that resveratrol uptake by hepatic cells involves two processes--a passive one and a carrier-mediated one. Thanks to this last process, resveratrol, while tightly bound to blood proteins, could be largely delivered to body tissues. The intracellular proteic targets of resveratrol remain to be identified.

This work reports significant advances on the transport in hepatic cells of resveratrol, a natural polyphenol with potent protective properties. First, we describe a new simple technique to qualitatively follow resveratrol cell uptake and intracellular distribution, based on resveratrol fluorescent properties. Second, the time-course study and the quantification of (3)H-labelled resveratrol uptake have been performed using human hepatic derived cells (HepG2 tumor cells) and hepatocytes. The temperature-dependence of the kinetics of uptake as well as the cis-inhibition experiments agree with the involvement of a carrier-mediated transport in addition to passive diffusion. The decrease of passive uptake resulting from resveratrol binding to serum proteins brings to light a mediated mechanism in physiological situation.

This paper reviews aspects concerning the genetic regulation of the expression of the well studied peroxisomal genes including those of fatty acid beta-oxidation enzymes; acyl-CoA oxidase, multifunctional enzyme and thiolase from different tissues and species. An important statement is PPARalpha, which is now long known to be in rodents the key nuclear receptor orchestrating liver peroxisome proliferation and enhanced peroxisomal beta-oxidation, does not appear to control so strongly in man the expression of genes involved in peroxisomal fatty acid beta-oxidation related enzymes. In this respect, the present review strengthens among others the emerging concept that, in the humans, the main genes whose expression is up-regulated by PPARalpha are mitochondrial and less peroxisomal genes. A special emphasis is also made on the animal cold adaptation and on need for sustained study of peroxisomal enzymes and genes; challenging that some essential roles of peroxisomes in cell function and regulation still remain to be discovered.

d -β-Hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30), a membrane enzyme, has been purified to homogeneity from dromedary ( Camelus dromedarius ) liver mitochondria. Our new purification method consisted of the solubilization of mitochondrial membranes by Triton X 100 and purification of BDH by two steps: DEAE-Sephacel and Phenyl-Sepharose. The molecular mass of the enzyme subunit size was 67 kDa. The purified enzyme is recognized by anti rat liver mitochondrial BDH antibodies. Furthermore, BDH activity was absolutely dependent upon phospholipids. BDH is also characterized by specific enzymatic parameters: an optimum pH of approximately 8 for the oxidation reaction, and approximately 7 for the reduction reaction and kinetic constant (Michaelis and dissociation constants) values of 1.07±0.13 mM for K MBOH , 0.21±0.01 mM for K MNAD + , 1.04±0.20 mM for K DNAD + , 0.29±0.01 mM for K MAcAc , 0.27±0.03 mM K MNADH and 1.12±0.18 mM for K DNADH .

International audience; In this study, we aimed to evaluate the antioxidant and anti-inflammatory properties of Opuntia ficus-indica cactus cladode extracts in microglia BV-2 cells. Inflammation associated with microglia activation in neuronal injury can be achieved by LPS exposure. Using four different structurally and biologically well-characterized LPS serotypes, we revealed a structure-related differential effect of LPS on fatty acid β-oxidation and antioxidant enzymes in peroxisomes: Escherichia coli-LPS decreased ACOX1 activity while Salmonella minnesota-LPS reduced only catalase activity. Different cactus cladode extracts showed an antioxidant effect through microglial catalase activity activation and an anti-inflammatory effect by reducing nitric oxide (NO) LPS-dependent production. These results suggest that cactus extracts may possess a neuroprotective activity through the induction of peroxisomal antioxidant activity and the inhibition of NO production by activated microglial cells.

International audience; The objective of this study was to evaluate the biological activities of the major phytosterols present in argan oil (AO) and in cactus seed oil (CSO) in BV2 microglial cells. Accordingly, we first determined the sterol composition of AO and CSO, showing the presence of Schottenol and Spinasterol as major sterols in AO. While in CSO, in addition to these two sterols, we found mainly another sterol, the Sitosterol. The chemical synthesis of Schottenol and Spinasterol was performed. Our results showed that these two phytosterols, as well as sterol extracts from AO or CSO, are not toxic to microglial BV2 cells. However, treatments by these phytosterols impact the mitochondrial membrane potential. Furthermore, both Schottenol and Spinasterol can modulate the gene expression of two nuclear receptors, liver X receptor (LXR)-alpha and LXR beta, their target genes ABCA1 and ABCG1. Nonetheless, only Schottenol exhibited a differential activation vis-a-vis the nuclear receptor LXR beta. Thus Schottenol and Spinasterol can be considered as new LXR agonists, which may play protective roles by the modulation of cholesterol metabolism.

The enzymes required for the beta-oxidation of fatty acyl-CoA are present in peroxisomes and mitochondria. Administration of hypolipidaemic compounds such as clofibrate to rodents leads to an increase in the volume and density of peroxisomes in liver cells. These proliferators also induce simultaneously the expression of genes encoding acyl-CoA oxidase, enoyl-CoA hydratase-hydroxyacyl-CoA dehydrogenase (multifunctional enzyme) and thiolase (3-ketoacyl-CoA thiolase). All these enzymes are responsible for long-chain and very-long-chain fatty acid beta-oxidation in peroxisomes. Similar results were observed when rat hepatocytes, or liver-derived cell lines, were cultured with a peroxisome proliferator. The increased expression of these genes is due to the stimulation of their transcription rate. These results show that the peroxisome proliferators act on the hepatic cells and regulate the transcription through various cellular components and pathways, including peroxisome-proliferator-activated receptor alpha (PPARalpha). After activation by specific ligands, either fibrates or fatty acid derivatives, PPARalpha binds to a DNA response element: peroxisome-proliferator-responsive element (PPRE), which is a direct repeat of the following consensus sequence: TGACCTXTGACCT, found in the promoter region of the target genes. PPARalpha is expressed mainly in liver, intestine and kidney. PPARalpha is a transcriptional factor, which requires other nuclear proteins for function including retinoic acid X receptor (RXRalpha) and other regulatory proteins. From our results and others we suggest the role of PPARalpha in the regulation of the peroxisomal fatty acid beta-oxidation. In this regard, we showed that although PPARalpha binds to thiolase B gene promoter at -681 to -669, a better response is observed with hepatic nuclear factor 4 ("HNf-4"). Moreover, rat liver PPARalpha regulatory activity is dependent on its phosphorylated state. In contrast, a protein-kinase-C-mediated signal transduction pathway seems to be modified by peroxisome proliferators, leading to an increase in the phosphorylation level of specific proteins, some of which have been shown to be involved in the phosphoinositide metabolism.

The first PPAR (peroxisome proliferator-activated receptor) was cloned in 1990 by Issemann and Green (Nature 347:645-650). This nuclear receptor was so named since it is activated by peroxisome proliferators including several drugs of the fibrate family, plasticizers, and herbicides. This receptor belongs to the steroid receptor superfamily. After activation by a specific ligand, it binds to a DNA response element, PPRE (peroxisome proliferator response element), which is a DR-1 direct repeat of the consensus sequence TGACCT x TGACCT. This mechanism leads to the transcriptional activation of target genes (Motojima et al., J Biol Chem 273:16710-16714, 1998). After the first discovery, several isoforms were characterized in most of the vertebrates investigated. PPAR alpha, activated by hypolipidemic agents of the fibrate family or by leukotrienes; regulates lipid metabolism as well as the detoxifying enzyme-encoding genes. PPAR beta/delta, which is not very well known yet, appears to be more specifically activated by fatty acids. PPAR gamma (subisoforms 1, 2, 3) is activated by the prostaglandin PGJ2 or by antidiabetic thiazolidinediones (Vamecq and Latruffe, Lancet 354:411-418, 1999). This latter isoform is involved in adipogenesis. The level of PPAR expression is largely dependent on the tissue type. PPAR alpha is mainly expressed in liver and kidney, while PPAR beta/delta is almost constitutively expressed. In contrast, PPAR gamma is largely expressed in white adipose tissue. PPAR is a transcriptional factor that requires other nuclear proteins in order to function, i.e. RXRalpha (9-cis-retinoic acid receptor alpha) in all cases in addition to other regulatory proteins. Peroxisomes are specific organelles for very long-chain and polyunsaturated fatty acid catabolism. From our results and those of others, the inventory of the role of PPAR alpha in the regulation of peroxisomal fatty acid beta-oxidation is presented. In relation to this, we showed that PPAR alpha activates peroxisomal beta-oxidation-encoding genes such as acyl-CoA oxidase, multifunctional protein, and thiolase (Bardot et al., FEBS Lett 360:183-186, 1995). Moreover, rat liver PPAR alpha regulatory activity is dependent on its phosphorylated state (Passilly et al., Biochem Pharmacol 58:1001-1008, 1999). On the other hand, some signal transduction pathways such as protein kinase C are modified by peroxisome proliferators that increase the phosphorylation level of some specific proteins (Passilly et al. Eur J Biochem 230:316-321, 1995). From all these findings, PPAR alpha and kinases appear to play an important role in lipid homeostasis.

Peroxisomes are present in most eukaryotic cell types, and have different enzymatic content and metabolic functions throughout the life scale. The endosymbiotic origin of these DNA-devoid organelles is supported by evolutionary data concerning genes encoding not only most peroxisomal proteins, but also several transcriptional factors regulating their expression such as peroxisome proliferator-activated receptors.

The effect of ciprofibrate on early events of signal transduction was previously studied in Fao cells. Protein kinase C (PKC) assays performed on permeabilized cells showed a more than two-fold increase in PKC activity in cells treated for 24 h with 500 microM ciprofibrate. To show the subsequent effect of this increase on protein phosphorylation, the in vitro phosphorylation on particulate fractions obtained from Fao cells was studied. Among several modifications, the phosphorylation of protein(s) with an apparent molecular mass of 85 kDa was investigated. This modification appeared in the first 24 h of treatment with 500 microM ciprofibrate. It was shown to occur on Ser/Thr residue(s). It was calcium but not calmodulin-dependent. The phosphorylation level of this/these protein(s) was reduced with kinase inhibitors and especially with 300 nM GF-109203X, a specific inhibitor of PKC. All these results suggest that the phosphorylation of the 85 kDa protein(s) is due to a PKC or to another Ser/Thr kinase activated via a PKC pathway. A possible biochemical candidate for 85 kDa protein seems to be the beta isoform of phosphatidylinositol 3-kinase regulatory subunit.

The basic mechanism(s) by which peroxisome proliferators activate peroxisome proliferator-activated receptors (PPARs) is (are) not yet fully understood. Given the diversity of peroxisome proliferators, several hypotheses of activation have been proposed. Among them is the notion that peroxisome proliferators could activate PPARs by changing their phosphorylation status. In fact, it is well known that several members of the nuclear hormone receptor superfamily are regulated by phosphorylation. In this report, we show that the rat Fao hepatic-derived cell line, known to respond to peroxisome proliferators, exhibited a high content of PPARalpha. Alkaline phosphatase treatment of Fao cell lysate as well as immunoprecipitation of PPARalpha from cells prelabeled with [32P] orthophosphate clearly showed that PPARalpha is indeed a phosphoprotein in vivo. Moreover, treatment of rat Fao cells with ciprofibrate, a peroxisome proliferator, increased the phosphorylation level of the PPARalpha. In addition, treatment of Fao cells with phosphatase inhibitors (okadaic acid and sodium orthovanadate) decreased the activity of ciprofibrate-induced peroxisomal acyl-coenzyme A oxidase, an enzyme encoded by a PPARalpha target gene. Our results suggest that the gene expression controlled by peroxisome proliferators could be mediated in part by a modulation of the PPARalpha effect via a modification of the phosphorylation level of this receptor.

The specific activity of carnitine palmitoyltransferase I (CPT-I) was similar in mitochondria isolated from rat Fao and human HepG2 hepatoma cells and from rat hepatocytes, but almost twofold higher in permeabilized hepatoma cells than in permeabilized hepatocytes. Short-term exposure to okadaic acid induced a ca. 80% stimulation of CPT-I in hepatocytes, whereas no significant response of the enzyme from hepatoma cells was evident. Thus, the high CPT-I activity displayed by hepatoma cells may be reached by hepatocytes upon challenge to okadaic acid. Reconstitution experiments with purified mitochondrial and cytoskeletal fractions showed that the cytoskeleton of hepatocytes produced a more remarkable inhibition of CPT-I than the cytoskeleton of Fao cells. The present data may be explained by a disruption of interactions between CPT-I and cytoskeletal components in tumor cells that may be involved in the okadaic acid-induced activation of hepatic CPT-I as previously suggested.

After our previous report on the cloning of two cDNA species in guinea pig, both encoding the same hepatic 79 kDa multifunctional protein 1 (MFP-1) [Caira, Cherkaoui-Malki, Hoefler and Latruffe (1996) FEBS Lett. 378, 57-60], here we report the cloning of a cDNA encoding a second multifunctional peroxisomal protein (MFP-2) in guinea-pig liver. This 2356 nt cDNA encodes a protein of 735 residues (79.7 kDa) whose sequence shows 83% identity with rat MFP-2 [Dieuaide-Noubhani, Novikov, Baumgart, Vanhooren, Fransen, Goethals, Vandekerckhove, Van Veldhoven and Mannaerts (1996) Eur. J. Biochem. 240, 660-666]. In parallel, we studied the effect of ciprofibrate, a hypolipaemic agent also known as peroxisome proliferator in rodent, on the expression of MFP-1 and MFP-2 (2.6 kb) in rats and guinea pigs. By Northern blotting analysis we demonstrated that three MFP-1-related mRNA species are expressed in the guinea-pig liver. The expression of two of them (3.5 and 2.6 kb) is slightly increased by ciprofibrate, whereas the 3.0 kb MFP-1 mRNA is, unlike the rat one, strongly down-regulated in guinea pigs treated with ciprofibrate. In a similar way, the hepatic expression of the guinea-pig 2.6 kb MFP-2 mRNA is also down-regulated in guinea pigs treated with ciprofibrate. These results demonstrate (1) that in contrast with the unique 3.0 kb MFP-1 rat mRNA, at least three hepatic MFP-1-related mRNA species are co-expressed in guinea pig; and (2) that, opposed to the accepted idea of non-responsiveness of the guinea pig to ciprofibrate, this drug affects MFP-1 and MFP-2 gene expression in this species. Also, the mRNA species for acyl-CoA oxidase and thiolase, two other enzymes of the peroxisomal β-oxidation pathway that are induced severalfold in responsive species are down-regulated in guinea pig. This paper is the first, to our knowledge, reporting the down-regulation of the expression of genes encoding enzymes involved in the peroxisomal β-oxidation of fatty acids (MFP-1) and bile acid synthesis (MFP-2) in mammals.

The structure of the rat liver's D-3-hydroxybutyrate dehydrogenase (BDH) active site has been investigated using an affinity alkylating reagent, the 3-chloroacetyl pyridine adenine dinucleotide (3-CAPAD). This NAD+ analogue reagent strongly inactivates the enzyme following a concentration- and time-dependent process with a stoichiometry of approximately 1. The reagent reacts at the coenzyme binding site as revealed by the efficient protection by NADH. The effect of 3-CAPAD is stronger with the enzyme into its natural membrane environment than with the lipid-free purified apoBDH or with the reconstituted apoBDH-mitochondrial phospholipid complex. The pH-dependent effect on the inactivation process is in agreement with the participation of protons in the catalytic mechanism of BDH. Furthermore, this study exhibits the phospholipid activating role in BDH catalytic activation.

Guinea pig was previously classified as a species nonresponsive to peroxisome proliferators. However, none of the previous reports was based on pharmacokinetic data. Here, after a comparative pharmacokinetic study between guinea pig and rat, we evaluate the guinea pig liver peroxisomal response to ciprofibrate, a hypolipemic agent and a potent peroxisome proliferator in rat. (1) Pharmacokinetic results show that plasmatic concentrations of ciprofibrate are equivalent in guinea pig and rat when guinea pigs are treated with ciprofibrate at 30 mg/kg twice a day and rats are treated at 3 mg/kg once a day. (2) The treatment of guinea pigs at 30 mg/kg twice a day for 2 weeks leads to a significant increase in the liver peroxisomal palmitoyl-CoA oxidase activity (×1.6) and also in the microsomal ω-laurate hydroxylase activity (×1.8). These increases are in accordance with the changes in polypeptide patterns of isolated liver peroxisomes as well as in the immunoblotting of acyl-CoA oxidase. It is deduced that a weak, but significant, peroxisome proliferation can occur in guinea pig liver after a ciprofibrate treatment at dosages corresponding to equivalent plasmatic concentrations of the drug between guinea pig and rat. (3) The hybridization of guinea pig liver RNA with the rat liver-inducible acyl-CoA oxidase cDNA probe shows a decrease in the corresponding heterologuous mRNA content after treatment with ciprofibrate at 30 mg/kg twice a day. This result contrasts with the slight increase observed in immunodetection and in enzymatic assays, suggesting the existence of at least two different acyl-CoA oxidases in guinea pig liver peroxisomes.

Flavanone, flavone, and tangeretin differentially affected the activities of cytochrome P540 1A and 2B isozymes in rat liver. Flavone and, to a lesser extent, tangeretin, increased activities of ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase, and pentoxyresorufin O-dealkylase (PROD), whereas flavanone mainly enhanced PROD activity. Immunoblot analysis indicated that flavone and tangeretin increased cytochrome P450 1A1, 1A2, and 2B1,2 forms, whereas flavanone only enhanced the cytochrome P450 2B isozymes. Northern blot study showed that flavone and tangeretin increased the level of the cytochrome P450 1A2 mRNAs. The concentration of the other mRNAs were slightly or not affected by flavonoids. These results suggest that the induction of P450 1A2 by flavone and tangeretin might involve a transcriptional and/or post-transcriptional mechanism.

The 2-enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD) is the second enzyme of the peroxisomal β-oxidation pathway. In human and rat, only one HD mRNA has been so far detected in the liver. This paper reports for the first time in a mammal species, the guinea pig, the cloning and sequencing of two cDNAs encoding an HD. The 3,274 nucleotide-cDNA is a strictly identical but longer copy of the 2,494 nucleotide-form. A 2,178 by-open reading frame encodes a protein of 726 amino acids ( M r 79.3 kDa) with the peroxisomal-targeting signal (tripeptide SKL) at the carboxyterminus. Northern blot analysis of HD mRNA identified three mRNAs of respective sizes 3.5, 2.6 and 1.6 kb in the guinea pig liver and kidneys.

The treatment of rats with ciprofibrate, a potent peroxisome proliferator, led to increased levels of the peroxisomal acyl-CoA oxidase (ACO) mRNA. How ciprofibrate functions to elevate ACO mRNA is not known. To help determine the mechanism of ciprofibrate action, in vitro transcription assays were performed. It was determined that ciprofibrate was responsible for a 3.5-fold stimulation of the rate of ACO transcription within 24 hr of ingestion. It was also observed that the transcription rate stimulation following a 2-week ciprofibrate treatment of Wistar rats was maintained following 4 weeks of ciprofibrate withdrawal. Re-introduction of the drug after the 4-week pause resulted in greater stimulation than was initially observed. The results demonstrate that the effect of ciprofibrate is rapid and persists at least twice as long as the initial treatment period. In Zucker rats, both lean and obese, ACO mRNA levels were examined following 2 weeks of ciprofibrate treatment (1 or 3 mg/kg body weight/day). The presence of increased blood levels of triglycerides did not increase ciprofibrate action on transcription, although basal levels of transcription of peroxisomal enzymes were higher in obese rats. The increase in the ACO mRNA level was greater than the transcription rate stimulation suggesting a post-transcriptional regulation.

Peroxisomes are ubiquitous organelles of eukaryotic cells and are present in significant amounts in hepatic liver cells. Peroxisomal enzymes contribute to several metabolic pathways including fatty acid, purine and amino acid catabolism or bile acid synthesis. The peroxisomal oxidative reactions produce hydrogen peroxide, mostly degraded by catalase which prevents oxidative stress. Moreover, peroxisomes are involved in arylderivative drug detoxification through its epoxide hydrolase activity.

Resveratrol is one of the most widely studied bio-active plant polyphenols. While its effect on endothelial blood vessel cells, cancer cells, inflammatory processes and neurodegenerative events is well documented, little is known about the implication of this phytophenol in differentiating processes, particularly in skeletal muscle cells. Here, we report the effects of resveratrol on mouse skeletal muscle-derived cells (C2C12) in either a nondifferentiated (myoblasts) or differentiated state (myotubes) by evaluating resveratrol uptake, cell proliferation, changes in cell shape, and the expression of genes encoding muscle-specific transcription factors or contractile proteins. Resveratrol: (1) rapidly accumulates within cells through passive and facilitated processes; (2) does not strongly affect cell viability, cell cycle and apoptosis; (3) behaves as a pro-differentiating agent as shown by the lengthening of cells, leading to a myotube phenotype; (4) upregulates muscular pro-differentiation markers and transcription factors (myogenin, Scrp3) starting after 12h of exposure and strongly increases heavy chain myosin content after 18h of exposure to resveratrol; (5) increases the Srf transcription factor's transcript level, a target mRNA of the miRNA-133b, which is itself downregulated by this polyphenol. These results put forward new pro-differentiating regulatory properties of resveratrol on skeletal muscles at least partly via modulation of specific miRNAs.

International audience; Peroxisomal 3-ketoacyl-CoA thiolase B (Thb) catalyzes the final step in the peroxisomal β-oxidation of straight-chain acyl-CoAs and is under the transcription control of the nuclear hormone receptor PPARα. PPARα binds to and is activated by the synthetic compound Wy14,643 (Wy). Here, we show that the magnitude of Wy-mediated induction of peroxisomal β-oxidation of radiolabeled (1-(14)C) palmitate was significantly reduced in mice deficient for Thb. In contrast, mitochondrial β-oxidation was unaltered in Thb(-/-) mice. Given that Wy-treatment induced Acox1 and MFP-1/-2 activity at a similar level in both genotypes, we concluded that the thiolase step alone was responsible for the reduced peroxisomal β-oxidation of fatty acids. Electron microscopic analysis and cytochemical localization of catalase indicated that peroxisome proliferation in the liver after Wy-treatment was normal in Thb(-/-) mice. Intriguingly, micro-array analysis revealed that mRNA levels of genes encoding cholesterol biosynthesis enzymes were upregulated by Wy in Wild-Type (WT) mice but not in Thb(-/-) mice, which was confirmed at the protein level for the selected genes. The non-induction of genes encoding cholesterol biosynthesis enzymes by Wy in Thb(-/-) mice appeared to be unrelated to defective SREBP-2 or PPARα signaling. No difference was observed in the plasma lathosterol/cholesterol ratio (a marker for de novo cholesterol biosynthesis) between Wy-treated WT and Thb(-/-) mice, suggesting functional compensation. Overall, we conclude that ThA and SCPx/SCP2 thiolases cannot fully compensate for the absence of ThB. In addition, our data indicate that ThB is involved in the regulation of genes encoding cholesterol biosynthesis enzymes in the liver, suggesting that the peroxisome could be a promising candidate for the correction of cholesterol imbalance in dyslipidemia.

Norbert Latruffe l ,I, Boubker Nasser ‘, Claude Morpain 3, Jiirgen Zirkel 4, Michael Seiter 4, Bernard Laude 3 and Wolfgang Trommer 4 ’ Laboratoire de Biobgie Mol&laire et Cellulaire, Universite’ de Bourgogne, Fact& des Sciences Mirande, BP 138, 21004 D@on Cedex (France) 2 Laboratoire de Biochimie &A CNRS 531) and 3 Laboratoire de Chimie Organique, Universite’ de Franche-Comte 25030 Besaqon Cedex (France) 4 Fachbereich Chemie, Universitiit Kaiserslautem, D 6750 Kaiserslautem (Germany)

Liver peroxisomes from three different species, rat, guinea pig and man, have been purified by ultracentrifugation on a discontinuous Nycodenz gradient. Several biochemical parameters were tested in order to compare the basic peroxisomal properties of liver from rat, a species strongly responsive to peroxisome proliferators, and guinea pig and man, two weakly responsive species. Polypeptide patterns were compared and the bands in guinea pig and man comigrating with the two major bands in rat, catalase at 66 kDa and urate oxidase at 35 kDa, appeared in low amounts. However, other polypeptides are similar throughout these species especially in guinea pig as revealed by cross-immunoreactivity using an anti-rat peroxisomal protein rabbit immune serum. Specific activities of peroxisome acyl-CoA oxidase and microsome omega-lauryl hydroxylase have comparable rates in rat and guinea pig liver, but in human liver the activities are much lower. There is a cross-hybridization between acyl-CoA oxidase mRNA probed by rat liver acyl-CoA oxidase cDNA among the three species at a medium stringency. But interestingly, acyl-CoA oxidase mRNA from guinea pig and man appear to be larger in size. On the other hand, the hormonal status does not seem to have a significant effect on the rat liver acyl-CoA oxidase mRNA level suggesting at most that insulin, corticosterone and estradiol have no direct effect on acyl-CoA oxidase gene expression, which contrasts with the well-known effect of peroxisome proliferators.

Cancers are the largest cause of mortality and morbidity in industrialized countries. In the field of the medicinal chemistry of natural products, numerous studies have reported interesting properties of trans-resveratrol as a chemopreventing agent against cancers, inflammation, and viral infection. Tumor growth inhibition has been linked to the ability of resveratrol to arrest cell cycle progression and to trigger cell death. This review focuses on the pathways that mediate resveratrol-induced cell death. Resveratrol impacts on the mitochondrial functions (respiratory chain, oncoproteins, gene expression, etc), in which p53 protein can be involved and its acetylated or phosphorylated forms. This polyphenol also affects death receptor distribution in ceramide-enriched membrane platforms which serve to trap and cluster receptor molecules, and facilitates the formation of a death-inducing signaling complex in the cell. To induce apoptosis, resveratrol also activates the ceramide / sphingomyelin pathway, which promotes ceramide generation and the downstream activation of kinase cascades. Resveratrol can activate alternative pathways to cell death such as those leading to autophagy, senescence or mitotic catastrophe. Furthermore, numerous attempts have been made using resveratrol analogs to improve the molecule's ability to block cell proliferation and induce cell death. Moreover, structural modification of natural phenolics is expected to produce analogs that may be useful tools to study the structure-activity relationships. Lastly, in various cancer types, resveratrol behaves as a chemosensitizer that lowers the threshold of cell death induction by classical anticancer agents and counteracts tumor cell chemoresistance.

International audience; Resveratrol (trans-3,4',5-trihydroxystilbene) is a natural antioxidant with cardiovascular and cancer preventive properties that is currently at the stage of pre-clinical studies for human cancer prevention. Beside its known effects on protein coding genes, one possible mechanism for resveratrol protective activities is by modulating the levels of non-coding RNAs. Here, we analyzed the effects of resveratrol on microRNA populations in human SW480 colon cancer cells. We establish that resveratrol treatment decreases the levels of several oncogenic microRNAs targeting genes encoding Dicer1, a cytoplasmic RNase III producing mature microRNAs from their immediate precursors, tumor-suppressor factors such as PDCD4 or PTEN, as well as key effectors of the TGFbeta signaling pathway, while increasing the levels of miR-663, a tumor-suppressor microRNA targeting TGFbeta1 transcripts. We also show that, while upregulating several components of the TGFbeta signaling pathway such as TGFbeta receptors type I (TGFbetaR1) and type II (TGFbetaR2), resveratrol decreases the transcriptional activity of SMADs, the main effectors of the canonical TGFbeta pathway. Our results establish that protective properties of resveratrol may arise at least in part from its capability to modify the composition of microRNA populations in cells, and suggest that the manipulation of the levels of key microRNAs, such as miR-663, may help to potentiate the anti-cancer and anti-metastatic effects of resveratrol.

International audience; To put forward BDH from Pseudomonas aeruginosa's enzymatic properties, we report a two-step purification of BDH and its gene sequencing allowing the investigation of its structural properties. Purification of BDH was achieved, using ammonium sulfate fractionation and Blue Sepharose CL-6B affinity chromatography. SDS-PAGE analysis reveals a MM of 29 kDa, whereas the native enzyme showed a MM of 120 kDa suggesting a homotetrameric structure. BDH encoding gene sequence shows a nucleotide open reading frame sequence of 771 bp encoding a 265 amino acid residues polypeptide chain. The modeling analysis of the three dimensional structure fits with the importance of amino acids in the catalysis reaction especially a strictly conserved tetrad. Amino-acid residues in interaction with the coenzyme NAD(+) were also identified.

International audience; Resveratrol is the subject of intense research because of the abundance of this compound in the human diet and as one of the most valuable natural chemopreventive agents. Further advances require new resveratrol analogs be used to identify the structural determinants of resveratrol for the inhibition potency of cell proliferation by comparing experimental and docking studies. Therefore, we synthesized new trans/(E)- and cis/(Z)-resveratrol - analogs not reported to date - by modifying the hydroxylation pattern of resveratrol and a double bond geometry. We included them in a larger panel of 14 molecules, including (Z)-3,5,4'-trimethoxystilbene, the most powerful molecule that is used as reference. Using a docking model complementary to experimental studies on the proliferation inhibition of the human colorectal tumor SW480 cell line, we show that methylation is the determinant substitution in inhibition efficacy, but only in molecules bearing a Z configuration. Most of the synthetic methylated derivatives (E or Z) stop mitosis at the M phase and lead to polyploid cells, while (E)-resveratrol inhibits cells at the S phase. Docking studies show that almost all of the docked structures of (Z)-polymethoxy isomers, but not most of the (E)-polymethoxy isomers substantially overlap the docked structure of combretastatin A-4, taken as reference ligand at the colchicine-tubulin binding site.

1. The properties of rat liver and bovine heart R-3-hydroxybutyrate dehydrogenase (BDH) have been extensively studied in the past 20 years, but little is known concerning the biogenesis and the regulation of this dehydrogenase over different species. 2. In addition, controversial results were often reported concerning the activity, the level and the subcellular location of this enzyme in ruminants. 3. BDH activity found in liver and kidney mitochondria from ruminants (cow and sheep) is low, while it is much higher in rat. 4. However, the enzyme activity is detected in microsomes and in cytosol of liver and of kidney cells from ruminants. These activities are not correlated to ketonaemia level. 5. Although low BDH activity is detected in liver mitochondria from ruminants; the bovine liver BDH gene seems to be translated since BDH can be immunodetected by using an antiserum raised against bovine heart BDH. 6. Beside this, the good cross-reactivity between heart BDH and liver BDH suggests their high level of homology in ruminants.

Peroxisome proliferation in rodent liver is a good biochemical marker of toxicology for several classes of xenobiotics, including fibrates; phthalates and adipates; or chlorophenoxy-acetate, a herbicide. Research in peroxisomes provides a good example of the integration of fields related to basic sciences and biomedical and industrial health. A 25 min video programme illustrates techniques involved in the characterization of purified peroxisomes ( Fig. 1 Download high-res image (70KB) Download full-size image Fig. 1 . Electron microscope view of isolated peroxisomes (from rat liver ×50 000). ) and membranes, immunoblotting, measurement of proliferation markers, mRNA analysis at the post-transcriptional level, DNA techniques, cell cultures as biological models and computer analysis. It is aimed at undergraduates and non-biochemist advanced students in biology.

International audience; Ciprofibrate is a well-known drug used to normalize lipid parameters and fibrinogen in atherosclerosis patients. In laboratory rodents such as rats or mice, ciprofibrate exhibits peroxisome proliferator activity. However, to date, no clear alterations or side effects caused by ciprofibrate have been noted in humans. In order to further investigate such possible relationships, we studied the effects of sustained ciprofibrate treatment in jerboas (Jaculus orientalis). In these rodents, ciprofibrate does not induce hepatomegaly or promote liver cell DNA replication, confirming that this species more closely resembles humans than do rats or mice. The jerboas were treated daily with ciprofibrate at 3 mg/kg body weight for 4 weeks. Subcellular markers, clinical enzymes and enzymatic antioxidant defenses were then assessed. The results showed a strong decrease in peroxisomal catalase activity and an increase in the level of malondialdehyde (a stress biomarker). Moreover, ciprofibrate in vivo and in vitro inhibited D-3-hydroxybutyrate dehydrogenase, a mitochondrial enzyme of the ketone body interconversion that is important in redox balance (NAD+/NADH+H+ ratio). In conclusion, under these conditions, ciprofibrate induced alterations in the liver oxidative metabolism.

International audience; Epidemiological studies suggested that trans-resveratrol, a wine grape component, could prevent malignant tumor development. This compound also demonstrated cytostatic and cytotoxic effects on tumor cells in vitro. To obtain trans-resveratrol derivatives with a better cellular uptake and enhanced antiproliferative effects, we synthesized a triacetate derivative as well as an oligomer, epsilon-viniferin and its acetylated form, epsilon-viniferin penta-acetate. We also obtained vineatrol, a wine grape shoot extract that associates several polyphenols that may act synergistically, including trans-resveratrol and epsilon-viniferin. We show here that resveratrol triacetate and vineatrol are as efficient as trans-resveratrol in inducing the accumulation of human colon cancer cells in early S phase of the cell cycle. This effect is associated with a nuclear redistribution of cyclin A and the formation of a cyclin A/cyclin-dependent kinase 2 complex whose kinase activity is increased. In contrast, epsilon-viniferin and its acetylated form do not demonstrate any significant activity on these cells when tested alone. Interestingly, resveratrol triacetate and vineatrol dramatically enhance 5-Fluoro-Uracil-mediated inhibition of colon cancer cell proliferation. Thus, acetylated derivatives of resveratrol have retained the cytostatic and cytotoxic activities of the parental molecule and thus deserve to be tested as chemosensitizers in animal models.