Martine Schmitt

Evolution of a Novel Phenolic Pathway for Pollen Development

From Retrogene to Phenolic Metabolism Metabolic plasticity, which involves the creation of new genes, is an essential feature of plant adaptation and speciation. Studying plants from the mustard family, Matsuno et al. (p. 1688) show that variants of the cytochrome P450 enzyme family were derived through retroposition, duplication, and subsequent mutaton. Evolutionary changes increased the volume of the substrate pocket altering with what sorts of substrates the enzymes could interact. The enzymes formed the basis for a new metabolic pathway, the products of which include constituents of pollen and of phenylpropanoid metabolism. Gene copying and positive Darwinian selection promoted the emergence of a phenolic pathway in Brassicaceae. Metabolic plasticity, which largely relies on the creation of new genes, is an essential feature of plant adaptation and speciation and has led to the evolution of large gene families. A typical example is provided by the diversification of the cytochrome P450 enzymes in plants. We describe here a retroposition, neofunctionalization, and duplication sequence that, via selective and local amino acid replacement, led to the evolution of a novel phenolic pathway in Brassicaceae. This pathway involves a cascade of six successive hydroxylations by two partially redundant cytochromes P450, leading to the formation of N1,N5-di(hydroxyferuloyl)-N10-sinapoylspermidine, a major pollen constituent and so-far-overlooked player in phenylpropanoid metabolism. This example shows how positive Darwinian selection can favor structured clusters of nonsynonymous substitutions that are needed for the transition of enzymes to new functions.

Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Analysis of the supramolecular organization of enzymes in the lignin pathway shows that cytochrome P450s oligomerize and move along with the very mobile plant endoplasmic reticulum. Their expression favors relocalization of their soluble partner proteins nearer the membrane and association of sequential enzymes in the pathway. Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.

Fluorescent Derivatives of the GFP Chromophore Give a New Insight into the GFP Fluorescence Process

The photophysical properties of synthetic compounds derived from the imidazolidinone chromophore of the green fluorescent protein were determined. Various electron-withdrawing or electron-donating substituents were introduced to mimic the effect of the chromophore surroundings in the protein. The absorption and emission spectra as well as the fluorescence quantum yields in dioxane and glycerol were shown to be highly dependent on the electronic properties of the substituents. We propose a kinetic scheme that takes into account the temperature-dependent twisting of the excited molecule. If the activation energy is low, the molecule most often undergoes an excited-state intramolecular twisting that leads it to the ground state through an avoided crossing between the S(1) and S(0) energy surfaces. For a high activation energy, the torsional motion within the compounds is limited and the ground-state recovery will occur preferentially by fluorescence emission. The excellent correlation between the fluorescence quantum yields and the calculated activation energies to torsion points to the above-mentioned avoided crossing as the main nonradiative deactivation channel in these compounds. Finally, our results are discussed with regard to the chromophore in green fluorescent protein and some of its mutants.

Anti-sedative and anti-cataleptic properties of NCS-382, a γ-hydroxybutyrate receptor antagonist

NCS-382 possesses antagonistic properties at gamma-hydroxybutyrate receptor sites. Its effect on the sedative/cataleptic behaviour observed in rats after gamma-hydroxybutyrate administration was investigated. NCS-382 diminished, in a dose-dependent manner, the sedative and/or cataleptic effects of gamma-hydroxybutyrate, as revealed by a variety of sensorimotor tests. These results indicate that the well-known sedative/anaesthetic effects induced by gamma-hydroxybutyrate administration are provoked via stimulation of a specific class(es) of gamma-hydroxybutyrate receptors which exist in the rat brain and which could mediate a local stimulation of opiate synthesis and release.

Effect of gamma-hydroxybutyrate and its antagonist NCS-382 on spontaneous cell firing in the prefrontal cortex of the rat

Gamma-hydroxybutyrate (GHB) at low doses (5-10 mg/kg i.p.) increased and at high doses (160-320 mg/kg i.p.) decreased the spontaneous firing rate of prefrontal cortex (PFC) neurons recorded in urethane-anesthetized rats. Only excitations were blocked by NCS-382, a specific GHB receptor antagonist; this suggests that the excitatory effect of low doses of GHB is mediated by a GHB receptor whereas the inhibitory effect of high doses of GHB involves a more complex mechanism.

Design, synthesis and analgesic properties of novel conformationally-restricted N-acylhydrazones (NAH).

A set of six azaheterocycles were designed as conformationally-constrained N-acylhydrazones and tested as analgesics.

Complexation with β-cyclodextrin confers oral activity on the flavonoid dioclein

Dioclein is a flavonoid reported to have many beneficial effects on the cardiovascular system such as vasorelaxant, hypotensive, antioxidant and antiarrythmogenic activities. However, use as pharmaceuticals is limited due to the lack of oral activity and low water solubility. In this work, intending to improve its oral activity, we performed a 1:1 inclusion complex (IC) between dioclein and beta-cyclodextrin (beta-CD). The IC was characterized by nuclear magnetic resonance and infrared spectroscopy and its vasodilator and hypotensive effects were evaluated in mice. The inclusion of dioclein in beta-CD increased the water solubility 44% compared to free dioclein. The IC (2.5mgkg(-1)) produced a higher and long lasting change in systolic blood pressure (SBP) after intraperitoneal administration compared to free dioclein. When given orally, free dioclein (10mgkg(-1)) showed no hypotensive effect while the IC induced a pronounced decrease in SBP. The in vitro vasodilator effect of dioclein was unchanged by its inclusion in beta-CD showing that the IC does not change the interaction between dioclein and its cellular targets. In conclusion, our results show that the new complex prepared by inclusion of dioclein in beta-CD improves the hypotensive effect of the flavonoid by increasing its bioavailability and enables dioclein to be effective after oral administration. The mechanism underling the increase in bioavailability is probably a consequence of a protective effect of beta-CD against in vivo biodegradation by enzymes and possibly increased water solubility.

Evidence for a role of high Km aldehyde reductase in the degradation of endogenous γ-hydroxybutyrate from rat brain

γ‐Hydroxybutyrate (GHB) is a putative neurotransmitter in brain. We have already demonstrated that it is transformed into γ‐aminobutyrate (GABA) by rat brain slices incubated under physiological conditions. This conversion occurs via a GABA‐transaminase reaction. Therefore, succinic semialdehyde, the oxidative derivative of GHB, appears to be the primary catabolite of GHB degradation. Apparently, the kinetic characteristics and pH optimum of GHB dehydrogenase (high K m, aldehyde reductase) in vitro do not favor a role for this enzyme in endogenous brain GHB oxidation. However, in the presence of glucuronate, glutamate, NADP and pyridoxal phosphate, pure GHB dehydrogenase, coupled to purified GABA‐transaminase does produce GABA from GHB at an optimum pH close to the physiological value and with a low K m for GHB.

Design and structure-activity relationship analysis of ligands of gamma-hydroxybutyric acid receptors

With the use of [3H]gamma-hydroxybutyric acid, binding experiments allowed the screening of new compounds as ligands of gamma-hydroxybutyric acid receptors. Starting from the acid-alcohol gamma-hydroxybutyric acid structure, structure-activity relation analysis and lead optimization highlighted gamma-hydroxybutyric acid derivatives with significantly increased affinities, when compared with the affinity of gamma-hydroxybutyric acid. Further pharmacological studies with the use of gamma-hydroxybutyric acid derivatives allowed the characterization of the first competitive antagonist acting at gamma-hydroxybutyric acid receptors (NCS 382).

New strategies towards proline derivatives as conformationally constrained arginine analogues

Abstract The cyano derivatives of 3-functionalized dehydroprolines led to arginine semi–constrained analogues.