Traitement des pathologies psychiatriques et stimulation magn��tique transcranienne r��p��t��e (rTMS): o�� en est-on ?

Objective

Over the past 15 years, the therapeutic effects of repetitive transcranial magnetic stimulation (rTMS) have been well studied in psychiatric disorders. We propose to conduct a qualitative review of the literature to allow a focus on the value of this technique, including its main indications.

Results

This technique, by the induction of a change in cortical excitability, can provide symptomatic improvement in various psychiatric disorders. We first focused on its effectiveness in mood disorders (mainly in uni or bipolar depressive episodes), then in schizophrenia (auditory hallucinations and negative symptoms), and lately in other psychiatric disorders. We are also considering how to implement this technique in clinical practice and future development.

Conclusion

Results reported in literature suggest that rTMS might fit into the strategies of caremainly for unipolar depression and auditory hallucinations. Recommendations should be published in this regard during 2011. Optimization of this technique and the extension to other psychiatric indications are still under study.     

Characterization of a chalcone synthase (CHS) flower-specific promoter from Lilium orential ��Sorbonne��

The first enzyme in the flavonoid pathway, chalcone synthase, is encoded by a gene (CHS) whose expression is normally under developmental control. In our previous studies, an 896-bp promoter region of a flower-specific CHS gene was isolated from Lilium orential ‘Sorbonne’, and designated as PLoCHS. Here, the PLoCHS promoter was fused to the β-glucuronidase (GUS) gene to characterize its spatial and temporal expression in Petunia hybrida ‘Dreams Midnight’ using an Agrobacterium-mediated leaf disc transformation method. Our results demonstrated that GUS expression was present in flowers, but reduced or absent in the other tissues (leaf and stem) examined. In petals, GUS activity reached its peak at flower developmental stage 4, and decreased at later stages. Deletion analysis indicated that even a 307-bp fragment of the PLoCHS promoter could still direct flower-specific expression. Further deletion of the region from −261 to −72 bp resulted in weak expression in different organs, including flowers, leaves and stems. This evidence combined with prediction of cis-acting elements in the PLoCHS promoter suggests that the TACPyAT box located in this promoter plays a key role in the regulation of organ-specific expression.     

Living wood fibers act as large-capacity ��single-use�� starch storage in black locust (Robinia pseudoacacia)

A living wood fiber (LWF) is one that retains the living protoplast. LWFs store numerous starch grains during the dormant period. In black locust (Robinia pseudoacacia), almost all wood fibers in the outer part of the annual ring are LWFs. In the outermost ring, starch is accumulated during the summer, retained in winter, and metabolized during spring. We determined the starch content of LWFs, ray parenchyma, and axial parenchyma using image analysis. More than 70% of the starch grains in the outermost ring were stored in LWFs during winter. After the breakdown of starch in spring, LWFs resulted in cell death. These results indicate that LWFs in black locust function as “single-use” large-capacity starch storage.     

Alcadein Cleavages by Amyloid ��-Precursor Protein (APP) ��- and ��-Secretases Generate Small Peptides, p3-Alcs, Indicating Alzheimer Disease-related ��-Secretase Dysfunction

Alcadeins (Alcs) constitute a family of neuronal type I membrane proteins, designated Alc_α, Alc_β, and Alc_γ. The Alcs express in neurons dominantly and largely colocalize with the Alzheimer amyloid precursor protein (APP) in the brain. Alcs and APP show an identical function as a cargo receptor of kinesin-1. Moreover, proteolytic processing of Alc proteins appears highly similar to that of APP. We found that APP α-secretases ADAM 10 and ADAM 17 primarily cleave Alc proteins and trigger the subsequent secondary intramembranous cleavage of Alc C-terminal fragments by a presenilin-dependent γ-secretase complex, thereby generating “APP p3-like” and non-aggregative Alc peptides (p3-Alcs). We determined the complete amino acid sequence of p3-Alc_α, p3-Alc_β, and p3-Alc_γ, whose major species comprise 35, 37, and 31 amino acids, respectively, in human cerebrospinal fluid. We demonstrate here that variant p3-Alc C termini are modulated by FAD-linked presenilin 1 mutations increasing minor β-amyloid species Aβ42, and these mutations alter the level of minor p3-Alc species. However, the magnitudes of C-terminal alteration of p3-Alc_α, p3-Alc_β, and p3-Alc_γ were not equivalent, suggesting that one type of γ-secretase dysfunction does not appear in the phenotype equivalently in the cleavage of type I membrane proteins. Because these C-terminal alterations are detectable in human cerebrospinal fluid, the use of a substrate panel, including Alcs and APP, may be effective to detect γ-secretase dysfunction in the prepathogenic state of Alzheimer disease subjects.     

Membrane Partitioning: ��Classical�� and ��Nonclassical�� Hydrophobic Effects

The free energy of transfer of nonpolar solutes from water to lipid bilayers is often dominated by a large negative enthalpy rather than the large positive entropy expected from the hydrophobic effect. This common observation has led to the idea that membrane partitioning is driven by the "nonclassical" hydrophobic effect. We examined this phenomenon by characterizing the partitioning of the well-studied peptide melittin using isothermal titration calorimetry (ITC) and circular dichroism (CD). We studied the temperature dependence of the entropic (-TΔS) and enthalpic (ΔH) components of free energy (ΔG) of partitioning of melittin into lipid membranes made of various mixtures of zwitterionic and anionic lipids. We found significant variations of the entropic and enthalpic components with temperature, lipid composition and vesicle size but only small changes in ΔG (entropy-enthalpy compensation). The heat capacity associated with partitioning had a large negative value of about -0.5 kcal mol(-1) K(-1). This hallmark of the hydrophobic effect was found to be independent of lipid composition. The measured heat capacity values were used to calculate the hydrophobic-effect free energy ΔG (hΦ), which we found to dominate melittin partitioning regardless of lipid composition. In the case of anionic membranes, additional free energy comes from coulombic attraction, which is characterized by a small effective peptide charge due to the lack of additivity of hydrophobic and electrostatic interactions in membrane interfaces [Ladokhin and White J Mol Biol 309:543-552, 2001]. Our results suggest that there is no need for a special effect-the nonclassical hydrophobic effect-to describe partitioning into lipid bilayers.     

Phosphorylation of Thr-516 and Ser-520 in the Kinase Activation Loop of MEKK3 Is Required for Lysophosphatidic Acid-mediated Optimal I��B Kinase �� (IKK��)/Nuclear Factor-��B (NF-��B) Activation

MEKK3 serves as a critical intermediate signaling molecule in lysophosphatidic acid-mediated nuclear factor-κB (NF-κB) activation. However, the precise regulation for MEKK3 activation at the molecular level is still not fully understood. Here we report the identification of two regulatory phosphorylation sites at Thr-516 and Ser-520 within the kinase activation loop that is essential for MEKK3-mediated IκB kinase β (IKKβ)/NF-κB activation. Substitution of these two residues with alanine abolished the ability of MEKK3 to activate IKKβ/NF-κB, whereas replacement with acidic residues rendered MEKK3 constitutively active. Furthermore, substitution of these two residues with alanine abolished the ability of MEKK3 to mediate lysophosphatidic acid-induced optimal IKKβ/NF-κB activation.     

Transforming Growth Factor-�� (TGF-��1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-�� Receptor Kinase Activity in Mesangial Cells

Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that signals through the interaction of type I (TβRI) and type II (TβRII) receptors to activate distinct intracellular pathways. TAK1 is a serine/threonine kinase that is rapidly activated by TGF-β1. However, the molecular mechanism of TAK1 activation is incompletely understood. Here, we propose a mechanism whereby TAK1 is activated by TGF-β1 in primary mouse mesangial cells. Under unstimulated conditions, endogenous TAK1 is stably associated with TβRI. TGF-β1 stimulation causes rapid dissociation from the receptor and induces TAK1 phosphorylation. Deletion mutant analysis indicates that the juxtamembrane region including the GS domain of TβRI is crucial for its interaction with TAK1. Both TβRI-mediated TAK1 phosphorylation and TGF-β1-induced TAK1 phosphorylation do not require kinase activity of TβRI. Moreover, TβRI-mediated TAK1 phosphorylation correlates with the degree of its association with TβRI and requires kinase activity of TAK1. TAB1 does not interact with TGF-β receptors, but TAB1 is indispensable for TGF-β1-induced TAK1 activation. We also show that TRAF6 and TAB2 are required for the interaction of TAK1 with TβRI and TGF-β1-induced TAK1 activation in mouse mesangial cells. Taken together, our data indicate that TGF-β1-induced interaction of TβRI and TβRII triggers dissociation of TAK1 from TβRI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation and not by the receptor kinase activity of TβRI.Members of the transforming growth factor-β (TGF-β)³ superfamily are key regulators of various biological processes such as cellular differentiation, proliferation, apoptosis, and wound healing (1, 2). TGF-β1, the prototype of TGF-β family, is a potent inducer of extracellular matrix synthesis and is well established as a central mediator in the final common pathway of fibrosis associated with progressive kidney diseases (3, 4). Upon ligand stimulation, TGF-β type I (TβRI) and type II (TβRII) receptors form heterotetrameric complexes, by which TβRI is phosphorylated in the GS domain and activated. Smad signaling pathway is well established as a canonical pathway induced by TGF-β1 (5, 6). Receptor-regulated Smads (Smad2 and Smad3) are recruited and activated by the activated TβRI. The phosphorylation in the GS domain (7) and L45 loop (8) of TβRI are crucial for its interaction with receptor-regulated Smads. After phosphorylation, receptor-regulated Smads are rapidly dissociated from TβRI and interact with common Smad (Smad4) followed by nuclear translocation. In addition to the Smad pathway, a recently emerging body of evidence has demonstrated that TGF-β1 also induces various Smad-independent signaling pathways (9–17) by which mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase (JNK) (18, 19), p38 MAPK (20–22), and extracellular signal-regulated kinase 1/2 (23, 24) can be activated by TGF-β1.TAK1, initially identified as a MAPK kinase kinase 7 (MKKK7 or MAP3K7) in the TGF-β signaling pathway (11, 12), also can be activated by environmental stress (25), proinflammatory cytokines such as IL-1 and TNF-α (26, 27) and lipopolysaccharide (28). For TAK1 activation, phosphorylation at Thr-187 and Ser-192 in the activation loop of TAK1 is essentially required (29–31). TAK1 can transduce signals to several downstream signaling cascades, including the MAPK kinase (MKK) 4/7-JNK cascade, MKK3/6-p38 MAPK cascade, and nuclear factor κB (NF-κB)-inducing kinase-IκB kinase cascade (26–28). A recent report has shown that TAK1 is also activated by agonists of AMP-activated kinase (AMPK) and ischemia, which in turn activates the LKB1/AMPK pathway, a pivotal energy-sensor pathway (32). TAK1 is also involved in Wnt signaling (33). We and others have previously demonstrated that TAK1 is a major mediator of TGF-β1-induced type I collagen and fibronectin expression through activation of the MKK3-p38 MAPK and MKK4-JNK signaling cascades, respectively (34–37). Furthermore, increased expression and activation of TAK1 enhance p38 phosphorylation and promote interstitial fibrosis in the myocardium from 9-day-old TAK1 transgenic mice (37). These data implicate a crucial role of TAK1 in extracellular matrix production and tissue fibrosis. TAK1 is also implicated in regulation of cell cycle (38), cell apoptosis (39–41), and the Smad signaling pathway (42–44). Thus, TAK1 may function as an important regulator and mediator of TGF-β1-induced Smad-dependent and Smad-independent signaling pathways.It has been demonstrated that TAK1 can be activated by the interaction with TAK1-binding protein 1 (TAB1) by in vitro binding assays and in overexpression studies (29–31); however, it is not clear whether TAB1 plays a crucial role in ligand-induced TAK1 activation. In embryonic fibroblasts from TAB1 null mice, IL-1 and TNF-α could induce TAK1-mediated NF-κB and JNK activation (45). TAK1 activation induced by TNF-α, IL-1, and T-cell receptor requires TAB2 or its homologous protein TAB3 (46–50). Although many questions still remain, much progress has been made in understanding the activation mechanism of TAK1 by inflammatory cytokines (46, 47, 51–53). Ligand binding of IL-1 receptor (IL-1R) results in recruitment of MyD88, which serves as an adaptor for IL-1 receptor-associated kinase (IRAK) 1 and 4. Subsequently IRAK1 is hyperphosphorylated and induces interaction with TNF-α receptor-associated factor 6 (TRAF6), resulting in TRAF6 oligomerization. After oligomerization of TRAF6, IRAK1-TRAF6 complex is dissociated from the receptor and associated with TAK1, which is mediated by TAB2 (or TAB3). In this process polyubiquitination of TRAF6 by Ubc13/Uev1A is thought to be critical for the association with TAB2 (or TAB3), which links TAK1 activation (46, 54, 55). In the case of TNF-α stimulation, TNF-α receptors form trimers and recruit adaptor proteins, TRAF2/5, and receptor-interacting protein 1 on the membrane. Ubc13/Uev1A- and TRAF2-dependent polyubiquitination of receptor-interacting protein 1 induce association of TAB2 (or TAB3), which then activates TAK1. Thus, TAB2 is required for ubiquitin-dependent activation of TAK1 by TRAFs. On the other hand, it has been demonstrated that hematopoietic progenitor kinase 1 plays a role as an upstream mediator of TGF-β-induced TAK1 activation, which in turn activates the MKK4-JNK signaling cascade in 293T cells (56, 57). Besides hematopoietic progenitor kinase 1, it has been also suggested that X-linked inhibitor of apoptosis (XIAP) might link TAK1 to TGF-β/BMP receptors through the capability of XIAP to interact with TGF-β/BMP receptors and TAB1 (58). Thus, although various molecules participate in the activation of TAK1, the precise mechanism by which TGF-β1 induces TAK1 activation is incompletely understood. Here, we provide evidence that the association of TAK1 with TGF-β receptors is important for TGF-β1-induced activation of TAK1 in mouse mesangial cells. TGF-β1 stimulation induces interaction of TβRI and TβRII, triggering dissociation of TAK1 from TβRI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation, independent of receptor kinase activity of TβRI.     

Cotton cultivation and textile production in the Arabian Peninsula during antiquity; the evidence from Mada��in Salih (Saudi Arabia) and Qal��at al-Bahrain (Bahrain)

The discovery of seeds and textiles from Gossypium (cotton) in Achaemenian levels of the mid-6th–late 4th century b.c. at Qal’at al-Bahrain, Bahrain and in early 1st millennium a.d. at Mada’in Salih, Saudi Arabia, reveals the role played by the Arabian Peninsula as a textile production centre during the centuries before and after the beginning of the Christian era. Both these sites were situated on important trade routes, overseas (Qal’at al-Bahrain) and overland (Mada’in Salih), and it is likely that at least part of the cotton production was intended for trade, complementing and perhaps competing with other sources of cotton textiles in the contemporary Middle East. In the arid climate of the Arabian Peninsula, cotton was probably grown in association with irrigated date palm gardens where a wide array of other crops was grown, as is shown by the analysis of charred seeds and wood from occupation levels at both sites. The present article places these particular finds in the larger context of cotton cultivation in the Middle East and India.     

MMP-9 Sheds the ��2 Integrin Subunit (CD18) from Macrophages

Activated macrophages are essential effectors of immunity and a rich source of matrix metalloproteinase-9 (MMP-9; gelatinase B). To search for cellular substrates of the enzyme, we subjected wild-type macrophages and macrophages expressing an autoactivating form of pro-MMP-9 (M9A macrophages) to proteomics analysis. Two-dimensional liquid chromatography together with tandem mass spectrometry identified 467 proteins in medium conditioned by M9A and/or wild-type macrophages. Subtractive proteomics identified 18 candidate MMP-9 substrates. Biochemical studies confirmed that two transmembrane proteins, β₂ integrin subunit (CD18) and amyloid protein precursor (APP), were enriched in the medium of M9A macrophages. To identify potential cleavage sites, we synthesized an overlapping library of peptides that spanned 60 residues of the ectodomain and transmembrane domain of β₂ integrin. Active MMP-9 cleaved a single peptide, ECVKGPNVAAIVGGT, at residues corresponding to Ala⁷⁰⁵ and Ile⁷⁰⁶ of the β₂ integrin. Peptides corresponding to this cleavage site were detected by tandem mass spectrometric analysis only in medium from M9A macrophages, strongly supporting the proposal that β₂ integrin is shed by autoactivating MMP-9. Our observations indicate that subtractive proteomics in concert with peptide substrate mapping is a powerful approach for identifying proteolytic substrates and suggest that MMP-9 plays previously unsuspected roles in the regulation and shedding of β₂ integrin.Matrix metalloproteinases (MMPs),¹ a subfamily of metazincins, are a structurally related group of zinc-dependent proteases (1). They are synthesized in latent form as pro-MMPs, and their prodomain must be removed or modified before they are proteolytically active. Some MMPs are secreted, whereas others are anchored to the cell surface, but their proteolytic activity is thought to be confined locally within the secretory pathway at the cell surface and nearby extracellular space (1–3). Individual MMPs have distinct substrate specificities and act on diverse extracellular and membrane proteins, such as chemokines, cell surface adhesion proteins, and extracellular matrix components. Proteolysis by MMPs plays an important role in a wide variety of normal and pathological processes, such as host defense, inflammation, and tumor progression (1–9).High levels of MMP-9 (gelatinase B) are expressed by activated macrophages (10), which are key effector cells of both innate and acquired immunity. In addition to having homeostatic functions, MMP-9 secreted by macrophages has been implicated in aneurysm formation, tumor progression, and disruption of atherosclerotic plaques (8, 9, 11, 12). Although the pathogenesis of those processes is generally thought to involve inappropriate degradation of extracellular matrix proteins, it has become increasingly clear that MMPs cleave a number of diverse substrates to mediate their varied functions (3, 13). Because MMP-9 can accumulate on the cell surface (14), it is likely to act on membrane proteins.To understand the specific roles of individual MMPs in inflammatory and immune responses, it is critical to identify their physiological substrates (3, 15–17). Most studies have focused on identifying substrates by their ability to be cleaved in defined in vitro reactions (18, 19), but this approach is biased in two ways. First, the candidate substrate must be selected a priori. Second, in vitro reactions fail to account for the complexity of the pericellular environment. Another method is to identify sequences in synthetic peptides that MMPs can cleave (20, 21). However, individual MMPs cleave different proteins at a variety of sites rather than at a consensus site. Moreover MMPs often interact with substrates through domains remote from the active site (exosites) (22), and exosites of MMP-2 have been used in a yeast two-hybrid system to trap candidate substrates (23). However, some substrates may bind weakly or not at all to exosites, limiting the utility of this approach for global substrate screening.An emerging strategy for finding MMP substrates is to conduct an unbiased, global search by coupling gel electrophoresis or liquid chromatography with MS-based protein identification. For example, two-dimensional (2D) gel electrophoresis (24) and derivatization of cysteine-containing peptides with an isotope affinity tag (25) have identified candidate substrates for membrane type-1 MMP (MT1-MMP) in plasma and cultured cells. Quantitative approaches using 2D difference gel electrophoresis have identified potential substrates of MMP-2 and MMP-9 in bronchoalveolar lavage fluid (26) and of MMP-9 and the related metalloproteinases ADAM-10 and ADAM-17 in cancer cells (27, 28). Lectin affinity chromatography detected glycosylated proteins that were selectively enriched in medium from a monocyte cell line expressing ADAM-17 and in phorbol ester-stimulated monocytes (16). Recently iTRAQ (isobaric tags for relative and absolute quantitation) labeling was used to identify substrates of MMP-2 (29). It is important to note, however, that proteases can affect protein abundance by pathways not involving proteolysis. Thus, an important limitation of many of these studies is that they fail to provide evidence that proteins with altered abundance in cells expressing a protease are direct substrates for proteolytic cleavage.In the current studies, we used subtractive proteomics to identify proteins enriched in the medium of a macrophage cell line. Subtractive proteomics compares two or more proteomes to identify proteins that are specifically enriched or depleted under certain conditions (30, 31). Our biochemical studies confirmed that two integral membrane proteins, amyloid precursor protein (APP) and the β₂ integrin subunit (CD18), were shed by macrophages expressing autoactivating MMP-9. We next used a peptide substrate mapping strategy to identify potential MMP-9 cleavage sites in β₂ integrin subunit. Targeted MS/MS analysis demonstrated that β₂ integrin subunit peptides with the same cleavage site were detected only in the medium of macrophages expressing autoactivating MMP-9, providing strong evidence that β₂ integrin is a direct substrate for proteolysis. Our observations indicate that subtractive proteomics in concert with peptide substrate mapping is a robust, high throughput technique for identifying cellular substrates that are proteolytically shed from macrophages.     

Select Small Core Structure Carbamates Exhibit High Contact Toxicity to ��Carbamate-Resistant�� Strain Malaria Mosquitoes, Anopheles gambiae (Akron)

Acetylcholinesterase (AChE) is a proven target for control of the malaria mosquito (Anopheles gambiae). Unfortunately, a single amino acid mutation (G119S) in An. gambiae AChE-1 (AgAChE) confers resistance to the AChE inhibitors currently approved by the World Health Organization for indoor residual spraying. In this report, we describe several carbamate inhibitors that potently inhibit G119S AgAChE and that are contact-toxic to carbamate-resistant An. gambiae. PCR-RFLP analysis was used to confirm that carbamate-susceptible G3 and carbamate-resistant Akron strains of An. gambiae carry wild-type (WT) and G119S AChE, respectively. G119S AgAChE was expressed and purified for the first time, and was shown to have only 3% of the turnover number (k _cat) of the WT enzyme. Twelve carbamates were then assayed for inhibition of these enzymes. High resistance ratios (>2,500-fold) were observed for carbamates bearing a benzene ring core, consistent with the carbamate-resistant phenotype of the G119S enzyme. Interestingly, resistance ratios for two oxime methylcarbamates, and for five pyrazol-4-yl methylcarbamates were found to be much lower (4- to 65-fold). The toxicities of these carbamates to live G3 and Akron strain An. gambiae were determined. As expected from the enzyme resistance ratios, carbamates bearing a benzene ring core showed low toxicity to Akron strain An. gambiae (LC₅₀>5,000 μg/mL). However, one oxime methylcarbamate (aldicarb) and five pyrazol-4-yl methylcarbamates (4a–e) showed good to excellent toxicity to the Akron strain (LC₅₀ = 32–650 μg/mL). These results suggest that appropriately functionalized “small-core” carbamates could function as a resistance-breaking anticholinesterase insecticides against the malaria mosquito.