PPARalpha transcriptionally induces AhR expression in Caco-2, but represses AhR pro-inflammatory effects.

In this work we demonstrate that Caco-2 cell treatment with WY-14643 (a potent PPARalpha agonist) causes an increase in AhR expression. Luciferase assays and directed mutagenesis experiments showed that induction mainly occurred at transcriptional level and involved a PPRE site located within the AhR promoter. These results were further confirmed by the use of PPARalpha knockout mice in which AhR induction by WY14643 was abrogated. In addition to CYP1 regulation, AhR has been described as being involved in inflammation, so we also studied the effect of AhR regulation by PPARalpha on the expression of some inflammation target genes. 3-Methylcholanthrene (a potent AhR agonist) increased the expression (mRNA) of the major inflammatory targets IL-1beta and MMP9. WY-14643 co-treatment abrogated the 3-methylcholanthrene pro-inflammatory effect. Hence the anti-inflammatory effect of PPARalpha overrides the pro-inflammatory effect of AhR.

Peroxidases have more functions than a Swiss army knife.

Plant peroxidases (class III peroxidases) are present in all land plants. They are members of a large multigenic family. Probably due to this high number of isoforms, and to a very heterogeneous regulation of their expression, plant peroxidases are involved in a broad range of physiological processes all along the plant life cycle. Due to two possible catalytic cycles, peroxidative and hydroxylic, peroxidases can generate reactive oxygen species (ROS) (*OH, HOO*), polymerise cell wall compounds, and regulate H2O2 levels. By modulating their activity and expression following internal and external stimuli, peroxidases are prevalent at every stage of plant growth, including the demands that the plant meets in stressful conditions. These multifunctional enzymes can build a rigid wall or produce ROS to make it more flexible; they can prevent biological and chemical attacks by raising physical barriers or by counterattacking with a large production of ROS; they can be involved in a more peaceful symbiosis. They are finally present from the first hours of a plant's life until its last moments. Although some functions look paradoxical, the whole process is probably regulated by a fine-tuning that has yet to be elucidated. This review will discuss the factors that can influence this delicate balance.

The novel non-glycosylated invertase from Candida utilis (the properties and the conditions of production and purification).

The Candida utilis yeast, which is cultivated in liquid media enriched with saccharose, synthesizes the well-known invertase of 300 kDa (EC 3.2.1.26). This enzyme is present both intracellularly in the periplasmic space and extracellularly in the culture broth. However, it was determined that the same C. utilis strain cultured in certain conditions is simultaneously capable of producing another, still unknown form of invertase with a molecular mass of 60 kDa. The presence of the latter enzymatic form was detected in cells as well as in the liquid culture medium. Both invertase forms were purified using a three-step process (ion-exchange chromatography, affinity chromatography, and preparative column electrophoresis) and named, due to their different migration ratio in polyacrylamide gel electrophoresis, F-form (Fast; 60 kDa) and S-form (Slow; 300 kDa). The F-form of invertase was found to be nonglycosylated as opposed to the well-known S-form of invertase from the same source. The physicochemical properties of the F-form of invertase (isoelectric point, substrate specificity, pH, and temperature optima) were determined and compared with those of the S-form of the enzyme.

Are hyperhydric shoots of Prunus avium L. energy deficient?

The content of oxidized and reduced pyridine nucleotides and some enzymatic activities of the oxidative pentose phosphate and glycolytic pathways were compared in normal (NS, growing on agar) and hyperhydric (HS, growing on gelrite) shoots of Prunus avium L. after 4 weeks of in vitro culture. The chlorophyll fluorescence from leaves and the redox capacity of the plasma membrane (reduction of exogenously added ferricyanide) of both types of shoots were recorded. The pool of oxidized and reduced pyridine nucleotides was lower in HS than in NS. These results suggested a reduced metabolism of HS in comparison to normal ones. This hypothesis was also supported by other observations. First, chlorophyll fluorescence measurements showed a lower chlorophyll content and a slight reduction of the photosynthetic capacity in HS. Second, the low activity of some enzymes of oxidative pentose phosphate pathway (OPP) and glycolysis indicated a decline of these biochemical pathways in HS with the consequence of a reduced production of chemical energy in the form of NAD(P)H and ATP. Finally, the lower reduction of ferricyanide by HS suggested a lower rate of redox reactions at the level of the plasma membrane of these shoots in comparison to NS.

Identification of a Ca(2+)-pectate binding site on an apoplastic peroxidase.

An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic acid in their Ca(2)+-induced conformation. By homology modeling, we were able to identify a motif of four clustered arginines (positions 117, 262, 268, and 271) that could be responsible for this binding. To verify the role of these arginine residues in the binding process, we prepared three mutants of APRX (M1, R117S; M2, R262Q/R268S; and M3, R262Q/R268S/R271Q). APRX and the three mutants were expressed as recombinant glycoproteins by the baculovirus-insect cell system. This procedure yielded four active enzymes with similar molecular masses that were tested for their ability to bind Ca(2)+-pectate. Recombinant wild-type APRX exhibited an affinity for the pectic structure comparable to that of the native plant isoperoxidase. The mutations impaired binding depending on the number of arginine residues that were replaced. M1 and M2 showed intermediate affinities, whereas M3 did not bind at all. This was demonstrated using an in vitro binding test and on cell walls of hypocotyl cross-sections. It can be concluded that APRX bears a Ca(2)+-pectate binding site formed by four clustered arginines. This site could ensure that APRX is properly positioned in cell walls, using unesterified domains of pectins as a scaffold.

Outside-in regulation of integrin clustering processes by ECM components per se and their involvement in actin cytoskeleton organization in a colon adenocarcinoma cell line.

We investigated in a colon adenocarcinoma cell line, the exclusive role of extracellular matrix (ECM) components in the absence of soluble factors regarding the integrin clustering processes, and their implication in cell adhesion, spreading and organization of the actin cytoskeleton. Caco-2 cells were shown to express at the plasma membrane 11 integrins, some of which (e.g. alpha3beta1, alpha5beta1, alpha6beta1/beta4, alpha8beta1 and alpha(v)beta1/beta5/beta6) were identified for the first time in this cell line. Cell adhesion and spreading processes were governed essentially by lamellipodium, the regulation of which was shown to be induced by two types of integrin clustering processes mediated by ECM proteins alone. During these phenomena, alpha2beta1, alpha(v)beta6 and alpha6beta1 integrins, the Caco-2 cell specific receptors of type IV collagen, fibronectin and laminin, respectively, were clustered in small focal complexes (point contacts), whereas alpha(v)beta5, the vitronectin receptor in this cell line, was aggregated in focal adhesions. The two levels of integrin clustering induced only F-actin cortical web formation organized in thin radial and/or circular filaments. We conclude thus that ECM components per se through their action on integrin clustering are involved in cell adhesion, cortical actin cytoskeleton organization and cell spreading.

Regulation of thyroid cell volumes and fluid transport: opposite effects of TSH and iodide on cultured cells.

Cell volume regulation by thyrotropin (TSH) and iodide, the main effectors involved in thyroid function, was studied in cultured thyroid cells. The mean cell volume, determined by performing 3-D reconstitution on confocal microscopy optical slices from living octadecylrhodamine-labeled cells cultured with both TSH and iodide (control cells), was 3.73 +/- 0.06 pl. The absence of iodide resulted in cell hypertrophy (136% of control value) and the absence of TSH in cell shrinkage (81%). These changes mainly affected the cell heights. The effect of TSH on cell volume was mediated by cAMP. The proportion of cytosolic volume (3-O-methyl-D-glucose space vs. total volume) decreased in the absence of iodide (85% of control value) and increased in the absence of TSH (139%), whereas protein content showed the opposite changes (121 and 58%, respectively). The net apical-to-basal fluid transport was also inversely controlled by the two effectors. Iodide thus antagonizes TSH effects on cell volumes and fluid transport, probably via adenylylcyclase downregulation mechanisms. The absence of either iodide or TSH may mimic the imbalance occurring in pathological thyroids.

Up-regulation of alpha 2 beta 1 integrin cell-surface expression protects A431 cells from epidermal growth factor-induced apoptosis.

High epidermal growth factor (EGF) concentration (10(-8) M) induces inhibition of A431 cell proliferation, resulting in part from an apoptotic process. For some cells escaping this process, proliferation was associated with a decrease in apoptosis. Moreover, these surviving cells displayed marked morphological changes consisting of filopodia formation and cell aggregation. Disrupting cell-cell contacts by lowering extracellular calcium concentration reversed the resistance process, suggesting that apoptosis protection by aggregation may involve intercellular adhesion and cell-cell survival signals probably mediated by calcium-requiring molecules such as integrins. From a panel of integrins tested, only alpha 2 beta 1 integrin cell-surface expression was up-regulated after high apoptotic EGF treatment, and this up-regulation was not observed under a growth-stimulatory EGF concentration (10(-11) M). Double-labeling analysis (alpha 2 beta 1/DNA) implicated alpha 2 beta 1 integrin in the resistance process since 99% of cells that up-regulated alpha 2 beta 1 integrin survived a high dose of EGF. Moreover, the involvement of alpha 2 beta 1 integrin up-regulation in the survival of A431 cells that escape EGF-induced apoptosis was verified using the blocking anti-alpha 2 beta 1 integrin antibody, which was shown to decrease the survival of EGF-stimulated cells. Furthermore, under our culture conditions, alpha 2 beta 1 integrin-dependent cell-cell adhesion can be inhibited without affecting other cell-adhesive interactions, suggesting that alpha 2 beta 1 integrin is involved more directly in cell-cell interaction than in cell-substrate adhesion. Our results provide evidence that EGF-induced up-regulation of alpha 2 beta 1 integrin contributes to the enhancement of cell-cell adhesion, leading to cell aggregate formation, which permits the escape of A431 cells to EGF-induced death by alpha 2 beta 1 integrin signaling.

NADH Oxidase Periodicity of Spinach Leaves Synchronized by Light.

The plasma membrane-associated NADH oxidase (NOX) of spinach leaf disks demonstrates a regular periodicity of 24.3+/-0.5 min (NADH = reduced nicotinamide adenine dinucleotide). Within a single population of plants, all exhibited a similar period. Experiments were carried out to determine what stimulus in the environment was responsible for this synchronization. The findings demonstrate that when plants are transferred from darkness to light, light exposure initiates a period maximum 12 min following the light exposure, independent of the point in the NOX cycle at which the light was given. Red light (650 nm, 10 min, 50 µM m-2 s-1) was equivalent to white light. Green or yellow light at a similar level as red light was ineffective. Far-red illumination (730 nm, 5 min) appeared to be equivalent to darkness and was sufficient to condition the plants to respond to light by resetting the NOX cycle. Involvement of phytochrome as a possible light-detecting molecule is indicated.

Molecular cloning and tissue-specific expression of an anionic peroxidase in zucchini.

A calcium-pectate-binding anionic isoperoxidase (APRX) from zucchini (Cucurbita pepo) was purified and subjected to N-terminal amino acid microsequencing. The cDNA encoding this enzyme was obtained by reverse transcriptase polymerase chain reaction from a cDNA library. It encoded a mature protein of 309 amino acids exhibiting all of the sequence characteristics of a plant peroxidase. Despite the presence of a C-terminal propeptide, APRX was found in the apoplast. APRX protein and mRNA were found in the root, hypocotyls, and cotyledons. In situ hybridization showed that the APRX-encoding gene was expressed in many different tissues. The strongest expression was observed in root epidermis and in some cells of the stele, in differentiating tracheary elements of hypocotyl, in the lower and upper epidermis, in the palisade parenchyma of cotyledons, and in lateral and adventitious root primordia. In the hypocotyl hook there was an asymmetric expression, with the inner part containing more transcripts than the outer part. Treatment with 2,3,5-triiodobenzoic acid reduced the expression of the APRX-encoding gene in the lower part of the hypocotyl. Our observations suggest that APRX could be involved in lignin formation and that the transcription of its gene was related to auxin level.

Shemin pathway and peroxidase deficiency in a fully habituated and fully heterotrophic non-organogenic sugarbeet callus: an adaptative strategy or the consequence of modified hormonal balances and sensitivities in these cancerous cells? A review and reassessment.

There are many arguments for considering a specific fully habituated (auxin and cytokinin-independent) and fully heterotrophic non-organogenic (HNO) sugarbeet callus cell line as terminating a neoplastic progression, and thus to be made of cancerous cells. The similarities with animal tumour and cancer cells are recalled. All types of habituated tissues examined in the literature share at least three common biochemical characteristics: low apparent peroxidase activity, high content of polyamines (PAs) and low production of ethylene. However, results concerning their auxin and cytokinin levels are not consistent. Peroxidase synthesis in the achlorophyllous HNO callus appears to arise from aminolevulinic acid (ALA) synthesis through the Shemin pathway, commonly used by animals and fungi. This pathway is limited by disturbed nitrogen metabolism that diverts glutamate (directly used for ALA synthesis in green higher plants) from the Kreb's cycle into PA synthesis. There is no argument to suggest that the low ethylene production is caused by a competition with PAs for their common precursor, S-adenosylmethionine. The results we report here indicate modified anabolic and catabolic pathways of auxins and cytokinins but also the possibilities of unusual compounds playing similar roles (dehydrodiconiferyl alcohol glucosides, for instance). A higher turnover of PAs is shown in the HNO callus, which could suggest a role for H2O2 and gamma-aminobutyric acid, products or intermediates in the PA catabolic pathway, as secondary messengers. The habituated cells retain some sensitivity towards exogenous auxins and cytokinins. Their increased sensitivity to PAs and ethylene suggests modified hormonal balances for the control of these actively dividing cells.

Thyrotropin chronically regulates the pool of thyroperoxidase and its intracellular distribution: a quantitative confocal microscopic study.

The regulation of thyroperoxidase (TPO) expression and of its intracellular distribution was studied in porcine thyroid cells cultured on porous bottom filters. Cells were cultured for 18 days in the absence or in the presence of thyrotropin (TSH) and with or without iodide. Microsomes were purified and analyzed by electrophoresis. TPO was detected by immunoblotting with polyclonal anti-porcine TPO antibodies and quantified by scanning the bands. The amount of TPO was increased 2-fold by TSH. High concentrations of iodide (1-50 microM, added daily) decreased the level of TPO. Confocal microscopy served to determine the intracellular localization of TPO and its quantitative distribution. Intracellular and surface-located TPO was detected by fluorescein-labeled antibodies on saponin-treated cells. Quantitative confocal microscopy showed that TSH increased the total amount of TPO 2-fold as for immunoblotting. The highest amount of TPO was found in the perinuclear area and between the nucleus and the Golgi apparatus. Only 4% of TPO was present on the apical surface and about 1% on the basolateral membrane; the remainder (about 95%) was inside the cells. TSH did not change these relative contents. TSH modified the intracellular distribution of the enzyme, increasing the TPO pool from the perinuclear area to apical membrane. This domain could be a site of storage of TPO. Adding a physiological concentration of iodide (0.5 microM, daily) did not influence the intracellular distribution of TPO. We concluded that chronic TSH stimulation 1) increased 2-fold the pool of TPO but did not change the relative proportion of TPO inside the cells and on the apical surface, and 2) modified the intracellular distribution of vesicular TPO, the major part of which was accumulated in the perinuclear and cytoplasmic area under the subapical domain of the polarized cells.

A minimal model for calcium signal generated by tyrosine kinase and G protein linked receptors; a stochastic computer simulation with CALSIM.

A software was designed to simulate the calcium signal following hormone or growth factor stimulation in epithelial cells. The software written in C runs on a PC under Windows environment. It is based on a Markov process where the dynamic of the system is characterised by phenomenological transition probabilities. Moreover a minimal model is proposed to analyse the role of plasma channels and IP3 receptors, together with the opposite action of the CaATPase pumps, in the cytosolic and endoplasmic reticulum (ER) calcium signal control. The simulation is applied on the calcium response following stimulation by carbacol (protein G coupled receptors) or epidermal growth factor (tyrosine kinase type receptors) in A431 epithelial cells. The experimental calcium signals can be grouped in three classes; a spike and a return to the basal level (signal A), a spike and a decrease to a plateau level (signal B) or a slow increase to a plateau (signal C). Epidermal growth factor induces signal A and B while carbacol gives signal B and C. When a 'pseudo' steady state is reached oscillations occur. Computer simulations show that signal A can result from the activation of IP3 receptors while signal C would result from the activation of the plasma channels; signal B appears as the additive contribution of both channels, while oscillations are compatible with a calcium induced calcium release mechanism. Simulations suggest that the calcium dynamic in the ER is a mirror of cytosolic calcium but that a simple way to produce similar calcium elevation in these two compartments is to activate plasma channels. Implications of such a mechanism is discussed.

Effects of a mechanical stimulation of localization of annexin-like proteins in Bryonia dioica internodes.

Mechanical stimulation exerted by rubbing a young internode of Bryonia dioica plants inhibits its growth. Previous cellular and biochemical studies showed that this growth inhibition is associated with Ca(2+) redistribution and profound modifications of plasma membrane characteristics. We extracted and purified Ca(2+)-dependent phospholipid-binding proteins from B. dioica internodes. Two main proteins, p33 and p35, and other minor bands were isolated and identified as annexin-like proteins because of their biochemical properties and their cross-reactions with antibodies against maize (Zea mays L.) annexins. Rabbit antiserum was obtained by injection of B. dioica p35. This antiserum was used for the immunocytolocalization of annexin-like proteins in internode parenchyma cells. It appeared that the distribution of annexin-like proteins was different before and 30 min after the mechanical stimulation. Western analysis of proteins in membrane fractions after separation by free-flow electrophoresis showed that p35 was present in most fractions, whereas p33 appeared mainly in plasmalemma-enriched fractions after the mechanical stimulation. It is hypothesized that a subcellular redistribution of these proteins might be involved in growth inhibition by mechanical stress.

Peroxidase activity in shoot apical meristem from Spinacia.

Peroxidase activity was localized in the shoot apical meristem (SAM) of Spinacia at the light and electron microscope level with the histochemical method employing H2O2 and 3,3'-diaminobenzidine. At the light microscopic level, peroxidase activity was examined in unfixed cryostat sections at different pHs. The enzyme was found to be more intense at low than at neutral and high pH. The activity was evenly distributed over the different regions of the meristem at low pH, whereas it was more intense in the rib meristem at pH 6.8 and 7.2. At the ultrastructural level, peroxidase activity was found at 3 cellular sites: the cell wall, the tonoplast and the plasmalemma. These results were discussed in relationship with the in situ distribution of peroxidases in plant cells and other enzymes in vegetative SAM.

The hormone-responsive NADH oxidase of the plant plasma membrane has properties of a NADH:protein disulfide reductase.

Plasma membranes of plant cells are characterized by a plant hormone (auxin)-responsive oxidation of NADH. The latter proceeds under argon. Also, when NADH oxidation is stimulated 50% by auxin addition, oxygen consumption is reduced by 40%. These findings are reconciled by direct assays using 5,5'-dithiobis-(2nitrobenzoic acid) (DTNB) (Ellman's reagent) that show protein disulfides to be electron acceptors for auxin-stimulated NADH oxidation. In the presence of an external reducing agent such as NADH, cysteine, or dithiothreitol, protein disulfides of the membrane are reduced with a concomitant stoichiometric increase in free thiols. In the absence of an external reducing agent, or in the presence of oxidized glutathione, DTNB-reactive thiols of the plasma membrane are decreased in the presence of auxins. Several auxin-reductant combinations were effective, but the same reductants plus chemically related and growth-inactive auxin analogs were not. A cell surface location of the affected thiols demonstrated with detergents and impermeant thiol reagents suggests that the protein may have a different physiological role than oxidation of NADH. For example, it may carry out some other role more closely related to the function of the auxin hormones in cell enlargement such as protein disulfide-thiol interchange.

Distribution of estrogen receptor heterogeneity in growing MCF-7 cells measured by quantitative microscopy.

The existence of interactive subpopulations is a biological feature that can modulate the proliferation of tumor cells. The hormone-responsive breast cancer cell line MCF-7 has been described as heterogeneous in terms of density. In this study we describe a quantitative image analysis methodology that we developed for the in situ detection of different subpopulations in MCF-7 cell cultures. Using this technology, we demonstrate the heterogeneity of the MCF-7 cell line in terms of both nuclear size and estrogen-receptor content. Analysis of the organization (topography) of the different subpopulations in culture reveals a nonrandom distribution of cells. When studying the development of these cell subpopulations as a function of time of culture, we observe modifications of their topography associated with an increase of estrogen-receptor-expressing cells. Moreover, the use of cluster analysis allows study of the local organization of these subpopulations. These changes appear to be independent of cell proliferation.

Measuring Beam Sizes and Ultra-Small Electron Emittances Using an X-ray Pinhole Camera.

A very simple pinhole camera set-up has been built to diagnose the electron beam emittance of the ESRF. The pinhole is placed in the air next to an Al window. An image is obtained with a CCD camera imaging a fluorescent screen. The emittance is deduced from the size of the image. The relationship between the measured beam size and the electron beam emittance depends upon the lattice functions alpha, beta and eta, the screen resolution, pinhole size and photon beam divergence. The set-up is capable of measuring emittances as low as 5 pm rad and is presently routinely used as both an electron beam imaging device and an emittance diagnostic.

Thyrotropin regulation of basolateral Cl- and I- effluxes in thyroid follicles in culture.

This report describes chloride and iodide effluxes across the basolateral membrane of porcine thyroid follicles reconstituted in culture. Basolateral chloride efflux is activated by thyrotropin (TSH). TSH (10 mU/ml) induces a twofold increase in the initial rate of chloride efflux. Forskolin (FSK, 5 microM) which increases intracellular cAMP also stimulates the initial rate of chloride efflux 3.5-fold, whereas an increase in the free cytosolic Ca2+ with the ionophore A23187 or thapsigargin, fails to mimic the TSH effect. The chloride channel blocker 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB) dose dependently inhibits chloride efflux rates with the maximal and half maximal effects observed for 100 microM and 30 microM, respectively. Basolateral chloride efflux rates are also inhibited in the presence of the organic anion transporter blocker probenecid (5 mM) or the Cl-/HCO3- exchanger blocker 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS, 250 microM), respectively, by 60% and 40%, whereas it is not affected by ClO4 (100 microM). The initial rate of iodide efflux is weakly activated (1.4-fold) by TSH (10 mU/ml). TSH effect could be reproduced by agents known to activate Ca(2+)-dependent processes as A23187, ionomycin (1 microM), phorbol 12-myristate 13-acetate (TPA, 0.1 microM) and epidermal growth factor (EGF, 0.1 microM) which increase the initial rate of iodide efflux from 1.2- to 1.8-fold, whereas FSK is without effect. The chloride channel blocker NPPB (500 microM) is required to significantly inhibit the initial rate of iodide efflux by 30%. The initial rate of iodide efflux is also reduced by 30% in the presence of SITS (250 microM) or probenecid (5 mM) whereas it is activated by ClO4 (100 microM). We conclude that basolateral chloride and iodide effluxes are both regulated by TSH, using two different transduction pathways. Chloride efflux regulation may involve a cAMP transduction signal, whereas the regulation of iodide efflux may involve a Ca2+ signal. Furthermore, as the sensitivities of chloride and iodide effluxes for the anion transporter blockers (especially NPPB) are different, it seems likely that chloride and iodide use two different transport pathways.

Chloride channel blockers inhibit the Na+/I- symporter in thyroid follicles in culture.

Porcine thyroid cells in culture are able to reorganize into well-polarized follicle-like structures in the presence of cAMP analogs. These follicles exhibit on their basolateral membrane domain the Na+/I- symporter which allows iodide to accumulate in the thyrocytes. The initial rate of iodide influx through the Na+/I- symporter is inhibited up to 98% by the chloride channel blockers. 5 nitro-2(3-phenylpropylamino)benzoic acid and 3',5-Dichlorodiphenylamine-2-carboxylic acid are the most effective inhibitors, with a K0.5 value of 60 microM. This inhibition is not secondary to inhibition of chloride transport. Other chloride transporter blockers have been studied but showed lesser activities.