Directeur de recherche émérite
Groupe thématique : Complexes métalliques et IRM, responsable du groupe
Hypoxia-inducing pathologies as cancer develop pathologic and inefficient angiogenesis which rules tumor facilitating microenvironment, a key target for therapy. As such, the putative ability of endothelial precursor cells (EPCs) to specifically home to hypoxic sites of neovascularization prompted to design optimized, site-specific, cell-mediated, drug-/gene-targeting approach. Thus, EPC lines were established from aorta-gonad-mesonephros (AGM) of murine 10.5 dpc and 11.5 dpc embryo when endothelial repertoire is completed. Lines representing early endothelial differentiation steps were selected : MAgEC10.5 and MagEC11.5. Distinct in maturation, they differently express VEGF receptors, VE-cadherin and chemokine/receptors. MAgEC11.5, more differentiated than MAgEC 10.5, displayed faster angiogenesis in vitro, different response to hypoxia and chemokines. Both MAgEC lines cooperated to tube-like formation with mature endothelial cells and invaded tumor spheroids through a vasculogenesis-like process. In vivo, both MAgEC-formed vessels established blood flow. Intravenously injected, both MAgECs invaded Matrigel(TM)-plugs and targeted tumors. Here we show that EPCs (MAgEC11.5) target tumor angiogenesis and allow local overexpression of hypoxia-driven soluble VEGF-receptor2 enabling drastic tumor growth reduction. We propose that such EPCs, able to target tumor angiogenesis, could act as therapeutic gene vehicles to inhibit tumor growth by vessel normalization resulting from tumor hypoxia alleviation.
Novel magneto-plasmonic nanoprobes were designed for multimodal diagnosis of cancer by combination of magnetic resonance imaging (MRI), surface-enhanced resonance Raman scattering (SERRS), and fluorescence emission in the very near infrared (VNIR). A controlled electrostatic assembly of silver nanoparticles (AgNPs), super-paramagnetic iron oxide nanopartides (SPIONs), VNIR dye Nile Blue (NB), and biopolymer chitosan (Chi) was used to formulate the AgIONs-Chi nanoprobes. The formulation protocol did not involve organic solvents and was rapid and efficient as confirmed by magnetic sorting. The SERRS response of the nanoprobes was very intense and constant for days. It decreased linearly upon 1000-fold dilution and was still recognizable at 0.1 nM NB concentration. After 30 days of storage, the SERRS loss was less than 30% and the hydrodynamic size of the AgIONs-Chi in PBS remained below 200 nm. The gradual decrease of the ratio SERRS/fluorescence allowed one to monitor the release of the fluorescent molecule upon long-term nanoprobe dissociation. The AgIONs-Chi exhibited 2-fold higher MRI contrast than that of commercially available SPION suspensions. Finally, the nanoprobes were actively uptaken by HeLa cancer cells and ensured trimodal MRI-SERRS-fluorescence detection of 10 itL cell inclusions in cm-sized agarose gels used here as phantom models of microtumors. The above results show that the magneto-plasmonic AgIONs-Chi are promising substrates for SERRS analysis in solution and for multimodal imaging of cancer cells.
We have developed new methods enabling in vivo localization and identification of metabolites through their (1)H NMR signatures, in a drosophila. Metabolic profiles in localized regions were obtained using HR-MAS Slice Localized Spectroscopy and Chemical Shift Imaging at high magnetic fields. These methods enabled measurement of metabolite contents in anatomic regions of the fly, demonstrated by a decrease in beta-alanine signals in the thorax of flies showing muscle degeneration.
L’imagerie par résonance magnétique (IRM) est maintenant entrée dans la vie courante, la moindre blessure aux sports d’hiver conduit à passer une IRM. Son caractère atraumatique et non invasif constitue un avantage décisif. La majorité des hôpitaux en sont équipés. L’IRM ne nécessite pas obligatoirement l’utilisation d’agents d’imagerie pour fournir une image structurelle de l’intérieur de l’organisme. Les réglages physiques de l’expérimentation permettent même d’accéder à différents paramètres des tissus (détection de tumeurs, oedèmes...). Ce n’est que si l’on veut aller plus loin dans la spécificité de la détection, qu’il est intéressant et même indispensable de faire appel à des agents de contraste injectables, même si l’on perd alors partiellement le caractère non invasif de l’IRM. Comme nous allons le montrer, la complexité des molécules va de l’agent chimiquement non spécifique jusqu’à l’agent de contraste dit « intelligent » (smart agent).
In the present study we investigated structural and metabolic modifications of the brain in the Ts65Dn mouse model of Down syndrome(DS)using both in vivo magnetic resonance imaging(MRI)and proton magnetic resonance spectroscopy(MRS). MRI was performed for further texture analysis and changes in texture parameters, including mean grey levels, contrast and homogeneity, and they were found in Ts65Dn compared to diploid littermates (2n). These phenotypic changes were different in the hippocampus and cerebellum, since in Ts65Dn mean grey levels increased in the cerebellum and decreased in the hippocampus. In addition, proton NMR spectra revealed differences in metabolite ratios. Levels of N-acetylaspartate(NAA)and glutamate(Glu), were lower compared to total creatine levels (CX), in the Ts65Dn brain. However, the most striking finding was an increase in the concentration of myo-inositol(Ins)and choline(Cho)in the hippocampus, whereas the Ins concentration was reduced in the cerebellum. Overall, these data illustrate that MRI and MRS are valuable assesment tools sufficiently sensitive to detect associated changes in different brain areas, thus providing new insight into the causative role of dosage-sensitive genes in the Ts65Dn DS mouse model.
PCP4/PEP19 is a modulator of Ca2 +-CaM signaling. In the brain, it is expressed in a very specific pattern in postmitotic neurons. In particular, Pcp4 is highly expressed in the Purkinje cell, the sole output neuron of the cerebellum. PCP4, located on human chromosome 21, is present in three copies in individuals with Down syndrome (DS). In a previous study using a transgenic mouse model (TgPCP4) to evaluate the consequences of 3 copies of this gene, we found that PCP4 overexpression induces precocious neuronal differentiation during mouse embryogenesis. Here, we report combined analyses of the cerebellum at postnatal stages (P14 and adult) in which we identified age-related molecular, electrophysiological, and behavioral alterations in the TgPCP4 mouse. While Pcp4 overexpression at P14 induces an earlier neuronal maturation, at adult stage it induces increase in cerebellar CaMK2alpha and in cerebellar LTD, as well as learning impairments. We therefore propose that PCP4 contributes significantly to the development of Down syndrome phenotypes through molecular and functional changes.
Cerebral malaria is a severe complication of Plasmodium falciparum infection. Although T-cell activation and type II IFN-gamma are required for Plasmodium berghei ANKA (PbA)-induced murine experimental cerebral malaria (ECM), the role of type I IFN-alpha/beta in ECM development remains unclear. Here, we address the role of the IFN-alpha/beta pathway in ECM devel-opment in response to hepatic or blood-stage PbA infection, using mice deficient for types I or II IFN receptors. While IFN-gammaR1(-)/(-) mice were fully resistant, IFNAR1(-)/(-) mice showed delayed and partial protection to ECM after PbA infection. ECM resistance in IFN-gammaR1(-)/(-) mice correlated with unaltered cerebral microcirculation and absence of ischemia, while WT and IFNAR1(-)/(-) mice developed distinct microvascular pathologies. ECM resistance appeared to be independent of parasitemia. Instead, key mediators of ECM were attenuated in the absence of IFNAR1, including PbA-induced brain sequestration of CXCR3(+)-activated CD8(+) T cells. This was associated with reduced expression of Granzyme B, IFN-gamma, IL-12Rbeta2, and T-cell-attracting chemokines CXCL9 and CXCL10 in IFNAR1(-)/(-) mice, more so in the absence of IFN-gammaR1. Therefore, the type I IFN-alpha/beta receptor pathway contributes to brain T-cell responses and microvascular pathology, although it is not as essential as IFN-gamma for the development of cerebral malaria upon hepatic or blood-stage PbA infection.
Tumor hypoxia is a characteristic of cancer cell growth and invasion, promoting angiogenesis, which facilitates metastasis. Oxygen delivery remains impaired because tumor vessels are anarchic and leaky, contributing to tumor cell dissemination. Counteracting hypoxia by normalizing tumor vessels in order to improve drug and radio therapy efficacy and avoid cancer stem-like cell selection is a highly challenging issue. We show here that inositol trispyrophosphate (ITPP) treatment stably increases oxygen tension and blood flow in melanoma and breast cancer syngeneic models. It suppresses hypoxia-inducible factors (HIFs) and proangiogenic/glycolysis genes and proteins cascade. It selectively activates the tumor suppressor phosphatase and tensin homolog (PTEN) in vitro and in vivo at the endothelial cell (EC) level thus inhibiting PI3K and reducing tumor AKT phosphorylation. These mechanisms normalize tumor vessels by EC reorganization, maturation, pericytes attraction, and lowering progenitor cells recruitment in the tumor. It strongly reduces vascular leakage, tumor growth, drug resistance, and metastasis. ITPP treatment avoids cancer stem-like cell selection, multidrug resistance (MDR) activation and efficiently enhances chemotherapeutic drugs activity. These data show that counteracting tumor hypoxia by stably restoring healthy vasculature is achieved by ITPP treatment, which opens new therapeutic options overcoming hypoxia-related limitations of antiangiogenesis-restricted therapies. By achieving long-term vessels normalization, ITPP should provide the adjuvant treatment required in order to overcome the subtle definition of therapeutic windows for in vivo treatments aimed by the current strategies against angiogenesis-dependent tumors.
In preclinical research, genetic studies have made considerable progress as a result of the development of transgenic animal models of human diseases. Consequently, there is now a need for higher resolution MRI to provide finer details for studies of small animals (rats, mice) or very small animals (insects). One way to address this issue is to work with high-magnetic-field spectrometers (dedicated to small animal imaging) with strong magnetic field gradients. It is also necessary to develop a complete methodology (transmit/receive coil, pulse sequence, fixing system, air supply, anesthesia capabilities, etc.). In this study, we developed noninvasive protocols, both in vitro and in vivo (from coil construction to image generation), for drosophila MRI at 9.4 T. The 10*10*80-μm resolution makes it possible to visualize whole drosophila (head, thorax, abdomen) and internal organs (ovaries, longitudinal and transverse muscles, bowel, proboscis, antennae and optical lobes). We also provide some results obtained with a Drosophila model of muscle degeneration. This opens the way for new applications of structural genetic modification studies using MRI of drosophila.
Single-walled carbon nanotubes (SWCNTs) containing traces of iron oxide were functionalized by noncovalent lipid-PEG or covalent carboxylic acid function to supply new efficient MRI contrast agents for in vitro and in vivo applications. Longitudinal (r1) and transversal (r2) water proton relaxivities were measured at 300 ?MHz, showing a stronger T2 feature as an MRI contrast agent (r2/r1 ?= ?190 for CO2H functionalisation). The r2 relaxivity was demonstrated to be correlated to the presence of iron oxide in the SWNT-carboxylic function COOH, in comparison to iron-free ones. Biodistribution studies on mice after a systemic injection showed a negative MRI contrast in liver, suggesting the presence of the nanotubes in this organ until 48 ?h after i.v. injection. The presence of carbon nanotubes in liver was confirmed after ex vivo carbon extraction. Finally, cytotoxicity studies showed no apparent effect owing to the presence of the carbon nanotubes. The functionalized carbon nanotubes were well tolerated by the animals at the dose of 10 ?mu g ?g-1 body weight.
A Th1 response is required for the development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM). The role of pro-Th1 IL-12 in malaria is complex and controversial. In this study, we addressed the role of IL-12R beta 2 in ECM development. C57BL/6 mice deficient for IL-12R beta 2, IL-12p40, or IL-12p35 were analyzed for ECM development after blood-stage PbA infection in terms of ischemia and blood flow by noninvasive magnetic resonance imaging and angiography, T cell recruitment, and gene expression. Without IL-12R beta 2, no neurologic sign of ECM developed upon PbA infection. Although wildtype mice developed distinct brain microvascular pathology, ECM-resistant, IL-12R beta 2-deficient mice showed unaltered cerebral microcirculation and the absence of ischemia after PbA infection. In contrast, mice deficient for IL-12p40 or IL-12p35 were sensitive to ECM development. The resistance of IL-12R beta 2-deficient mice to ECM correlated with reduced recruitment of activated T cells and impaired overexpression of lymphotoxin-alpha, TNF-alpha, and IFN-gamma in the brain after PbA infection. Therefore, IL-12R beta 2 signaling is essential for ECM development but independent from IL-12p40 and IL-12p35. We document a novel link between IL-12R beta 2 and lymphotoxin-alpha, TNF-alpha, and IFN-gamma expression, key cytokines for ECM pathogenesis.
MRI, MRS and chemistry Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) are two techniques derived from NMR, which is well known by chemists. If MRI is the technique of choice for the non-invasive and atraumatic exploration of living beings, it is now moving towards obtaining additional information of biochemical nature, we can speak of "molecular imaging" and even of "imaging of gene expression". These developments require the creation of contrast agents specific of the enzymes of interest and that is where the chemistry is essential. In SRM, the role of the chemist is to transform high resolution NMR techniques to make them compatible with the very special characteristics of localized NMR of living environments. In this article, we will discuss the growing importance of chemistry in the spectacular development of MRI and MRS.
Gd(3)L is a trinuclear Gd(3+) complex of intermediate size, designed for contrast agent applications in high field magnetic resonance imaging (H(12)L is based on a trimethylbenzene core bearing three methylene-diethylenetriamine- N,N,N’’,N’’-tetraacetate moieties). Thanks to its appropriate size, the presence of two inner sphere water molecules and a fast water exchange, Gd(3)L has remarkable proton relaxivities at high magnetic field (r(1) = 10.2 vs 3.0 mM(-1) s(-1) for GdDOTA at 9.4 T, 37 degrees C, in H(2)O). Here we report an in vivo MRI feasibility study, complemented with dynamic gamma scintigraphic imaging and biodistribution experiments using the (153)Sm-enriched analog. MRI experiments were performed at 9.4 T in mice with Gd(3)L and the commercial contrast agent gadolinium(III)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (GdDOTA). Gd(3)L was well tolerated by the animals at the dose of 8 micromol Gd kg(-1) body weight. Dynamic contrast enhanced (DCE) images showed considerably higher signal enhancement in the kidney medulla and cortex after Gd(3)L injection than after GdDOTA injection at an identical dose. The relaxation rates, DeltaR(1), were calculated from the IR TrueFISP data. During the excretory phase, the DeltaR(1) for various tissues was similar for Gd(3)L and GdDOTA, when the latter was injected at a three-fold higher dose (24 vs 8 micromol Gd kg(-1) body weight). These results point to an approximately three times higher in vivo relaxivity (per Gd) for Gd(3)L relative to GdDOTA, thus the ratio of the relaxivities of the two compounds determined in vitro is retained under in vivo conditions. They also indicate that the two inner sphere water molecules per Gd in Gd(3)L are not substantially replaced by endogenous anions or other donor groups under physiological conditions. Gd(3)L has a pharmacokinetics typical of small, hydrophilic complexes, involving fast renal clearance and no retention in the blood pool. The dynamic gamma scintigraphic studies and the biodistribution experiments performed in Wistar rats with (153)Sm-enriched (*)Sm(3)L are also indicative of a fast elimination via the kidneys.
An amphiphilic gadolinium (III) chelate (GdL) was synthesized from commercially available stearic acid. Aqueous solutions of the complex at different concentrations (from 1 mM to 1 mu M) were prepared and adsorbed on multiwalled carbon nanotubes. The resulting suspensions were stable for several days and have been characterized with regard to magnetic resonance imaging (MRI) contrast agent applications. Longitudinal water proton relaxivities, r(1), have been measured at 20, 300, and 500 MHz. The r(1) values show a strong dependence on the GdL concentration, particularly at low field.
A yeast artificial chromosome (YAC) transgenic murine model of partial trisomy 21 overexpressing five human genes-including DYRK1A, which encodes a serine threonine kinase involved in cell cycle control-has been shown to present an increase in brain weight. We analyzed this new phenotype by measuring total and regional brain volumes at different ages, using a 7 Tesla magnetic resonance imaging volumetric approach. Volumetric measurements showed a total volume increase of 13.6% in adult mice. Changes in brain morphogenesis were already visible at a very early postnatal stage (postnatal days 2-7). Region-specific changes were characterized from postnatal day 15 to 5 months. These results, made it possible to define region-specific effects of DYRK1A overexpression, with the strongest increase seen in the thalamus-hypothalamus area (24%).
Background : The Kit gene encodes a receptor tyrosine kinase involved in various biological processes including melanogenesis, hematopoiesis and gametogenesis in mice and human. A large number of Kit mutants has been described so far showing the pleiotropic phenotypes associated with partial loss-of-function of the gene. Hypomorphic mutations can induce a light coat color phenotype while complete lack of KIT function interferes with embryogenesis. Interestingly several intermediate hypomorphic mutations induced in addition growth retardation and post-natal mortality.
Purpose : To asses the role of large b-value diffusion weighted imaging (DWI) in the characterization of the physicochemical properties ot the water in the brain edema under expeirmental and clinical conditions. Materials and Methods : Vasogenic brain edema was induced in mice by means of cold injury. A total of 17 patients wtih extensive peritumoral brain edema were also investigated. The longitudinal relaxation time(T-1) and apparent diffusion coefficient (D) were measured in the edematous area both in humans and in mice. D was calculated by using both mono- (D-mono) and biexponential (D-fast an D-slow) approaches in the low and overall range of b-values, respectively. The D values were correlated with the T-1 values. Results : A strong linear correlation was found between T-1 and D-mono in vasogenic brain edema, both in humans and in mice. After breakdown of D-mono into fast and slow diffusing components, only Dfast exhibited a strong correlation with T-1 : D-slow was unchanged in vasogenic brain edema. Conclusion : Large b-value DWI can furnish a detailed characterization of vasogenic brain edema, and may provide a quantitative approach for the differentiation of edema types on the basis of the physicochemical properties of the water molecules. Application of the DWI method may permit prediction and follow-up of the effects of antiedematous therapy.
Recent studies have shown that cell migration can be monitored in vivo by magnetic resonance imaging after intracellular contrast agent incorporation. This is due to the dephasing effect on proton magnetization of the local magnetic field created by a labelled cell. Anionic iron oxide nanoparticles (AMNP) are among the most efficient and non-toxic contrast agents to be spontaneously taken up by a wide variety of cells.
1-[6-(Acridine-9-carbonyloxy)hexyl]pyridinium chloride (1) was synthesized and studied as a potential inhibitor of acetylcholinesterase (AChE), which is frequently involved in Alzheimer’s disease. UV spectrophotometry showed that 1 is a reversible and competitive inhibitor of AChE (K-i approximate to 2 x 10(-7) M). NMR (TrNOESY) showed that 1, bonded to AChE, maintains an extended form that allows hydrophobic interactions to occur between the aliphatic chain and the deep and narrow gorge of AchE and favors interactions between the acridine group and the catalytic and anionic subsites situated at the bottom of the gorge, and also between the pyridinium ring and the peripheral site. A more detailed picture of the structure of the complex was obtained by combining NMR structural data and molecular modeling (docking, dynamics simulation and energy calculations). Copyright (c) 2006 John Wiley & Sons, Ltd.
In vivo imaging of small animals is a rapidly developing field. However, the potential of global imaging of infectious processes in animal models remains poorly explored. We used magnetic resonance imaging (MRI) to follow the development and regression of inflammatory lesions caused by infection by Klebsiella pneumoniae in mouse lungs. A virulent strain caused an intense inflammation within 2 days in the whole lungs, while an avirulent strain did not show significant changes. Mice infected with the virulent strain and subsequently treated with antibiotics presented a severe inflammation localized mainly in the left lung that disappeared after a week. The lesions observed by MRI correlated with the damage seen by histological analysis and a 3D representation of the tissue allowed better visualization of the development and healing of inflammatory lesions. MRI thus represents a powerful technique to study in vivo the interactions between a pathogen and its host in real time.
Magnetic resonance spectroscopy studies in animal models of prion disease are very few and concern terminal stages of infection. In order to study earlier stages of the disease, we used in vivo magnetic resonance spectroscopy in a mouse model of scrapie and, for the first time, in mice infected with a bovine spongiform encephalopathy strain. In bovine spongiform encephalopathy-infected mice, we observed an increase in myo-inositol preceding clinical signs by 20 days, followed by a decrease in N-acetylaspartate at advanced stages. In scrapie-infected mice, changes in N-acetylaspartate and myo-inositol were detected at the beginning of the symptomatic phase. These results show that magnetic resonance spectroscopy is a valuable tool for detecting subtle metabolic changes associated to gliosis and neuronal dysfunction in prion diseases.
We show here, for the first time, that two neutral polymers may completely associate together in water to spontaneously form supramolecular nanoassemblies (nanogels) of spherical shape. The cohesion of these stable structures of about 200 nm is based upon a "lock and key" mechanism : inclusion complexes are formed between the hydrophobic alkyl chains grafted on a polysaccharide (dextran) and the molecular cavities contained in a poly-cyclodextrin polymer. Production yields reached 95%. It was established that all the alkyl chains were included within the cyclodextrins’ cavities in these nanoassemblies. The multivalent character of the interactions between the two polymers ensures the stability of the nanoassemblies. Moreover, empty cyclodextrin units remained accessible for the inclusion of compounds of interest such as benzophenon or tamoxifen. (c) 2006 Elsevier B.V. All rights reserved.
It is demonstrated in this paper that an imaging spectrometer with the C-13 Magnetic Resonance Spectroscopy (MRS) technique is an original unintrusive and nondestructive method, perfectly adapted to determine the amount of CO2 dissolved in a closed bottle of Champagne or Sparkling wine as well as the self-diffusion of CO2 in this matrix. Two Champagnes and one Sparkling wine were analyzed on a 7T imaging spectrometer. It was necessary to use an imaging spectrometer with a large available bore to insert the bottle, localize the measurement and carry out H-1 and C-13 spectroscopy with a good sensitivity. In the C-13 spectra, CO2 and alcohol signals are well separated. By use of the quantity of ethanol acting as an internal reference, it was possible to deduce the CO2 concentration. We determined the relaxation parameter T-1 of CO2. Thanks to diffusion spectroscopy, the diffusion rate of the CO2 in the closed bottle was also calculated. The method can easily be extended to study the CO2 content in closed bottles of other sparkling drinks, like beers, sodas, and fizzy waters. (c) 2004 Elsevier B.V All rights reserved.
The in vivo spectrum of regenerating muscles shows a specific cross-correlation signal assigned to the (n-3) fatty acyl chain, which peaks during the myoblast fusion phase. In order to identify the origin of this signal and to take all the lipid metabolites into account, we investigated the degeneration-regeneration process by H-1 2D NMR of lipid muscle extracts. We observed an increase in the total amount of lipids during the regeneration process, although the lipid profile did not show any drastic change during this process. The changes in the NMR signal observed in vivo and, in particular, the appearance of the specific (n-3) fatty acyl chain signal appears to arise from mobile lipid compartments located in fusing cells. (c) 2005 Published by Elsevier B.V.
Localized in vivo NMR spectroscopy, chemical shift imaging or multi-voxel spectroscopy are potentially useful tools in small animals that are complementary to MRI, adding biochemical information to the mainly anatomical data provided by imaging of water protons. However the contribution of such methods remains hampered by the low spectral resolution of the in vivo I D spectra. Two-dimensional methods widely developed for in vitro studies have been proposed as suitable approaches to overcome these limitations in resolution.
It is generally believed that the apparent diffusion coefficient (ADC) changes measured by diffusion-weighted imaging (DWI) in brain pathologies are related to alterations in the water compartments. The aim of this study was to elucidate the role of compartmentalization in DWI via biexponential analysis of the signal decay due to diffusion. DWI experiments were performed on mouse brain over an extended range of b-values (up to 10000 mm(-2) s) under intact, global ischemic, and cold-injury conditions. DWI was additionally applied to centrifuged human erythrocyte samples with a negligible extracellular space. Biexponential signal decay was found to occur in the cortex of the intact mouse brain.
How the myocardium is able to permanently coordinate its intracellular fluxes of ATP synthesis, transfer and utilization is difficult to investigate in the whole organ due to the cellular complexity. The adult myocardium represents a paradigm of an energetically compartmented cell since 50% of total CK activity is bound in the vicinity of other enzymes ( myofibrillar sarcolemmal and sarcoplasmic reticulum ATPases as well as mitochondrial adenine nucleotide translocator, ANT). Such vicinity of enzymes is well known in vitro as well as in preparations of skinned fibers to influence the kinetic properties of these enzymes and thus the functioning of the subcellular organelles. Intracellular compartmentation has often been neglected in the NMR analysis of CK kinetics in the whole organ. It is indeed a methodological challenge to reveal subcellular kinetics in a working organ by a global approach such as NMR.
It is important to obtain high resolution images of joints for the study of disease, especially in rodent experimental models. We optimized H-1 magnetic resonance imaging three-dimensional sequences at 7 T, with lipid signal suppression, and T-1 and T-2 measurements for in vivo experiments on rat joints, in order to assess the effectiveness of high-field MRI. The method was validated by applying it to the early diagnosis of arthritis. We studied the progress of rheumatoid arthritis in an arthritic rat model. We observed the rats’ knees for 21 days after inducing arthritis. The images acquired over one hour had a high resolution of 1.75 x 10(-3) mm(3), (105 x 105 x 145 mum(3)) which allowed us to spot the early stages of joint degeneration, such as bone erosion, and to observe an apparent ’MRI’ loss of cartilage thickness, attributed to dehydration of the cartilage tissue. The MR images obtained during the early stages of rheumatoid arthritis enabled us to study joint changes accurately before any histological signs of attack were visible. Copyright (C) 2003 John Wiley Sons, Ltd.
Previous molecular modeling studies, in our laboratory, have shown that some esters of type RCOO(CH2)(n)C5H5N+Cl- are potentially active against Alzheimer’s disease. We have also demonstrated that acridine, which has strong anticholinesterase activity appears to be a suitable R substituent. The main obstacle to the possible pharmaceutical application of these compounds is their limited solubility in water, which is due to the poor aqueous solubility of acridine itself (0.26 mM). Inclusion complexation with cyclodextrins may overcome this problem.
Hepatic encephalopathy may occur following acute hepatic failure (AHF), which results in the release of toxic compounds from the injured liver. These compounds, which induce cerebral edema, are not well characterized, yet. The aim of this study was to evaluate the potential interest of NMR spectroscopy in the follow-up of different plasma compounds in pigs with ischemia-induced fulminant hepatic failure treated or not with a bioartificial liver (BAL), which has been previously shown to improve the neurological status of the animals.
The purpose of this study was to acquire a localized 2D (two-dimensional) H-1 correlation spectrum. in a volume of interest reasonably small, and within an experiment time compatible with clinical applications. A modified PRESS technique has been used. The last 180degrees pulse of the PRESS sequence has been converted into a 90degrees pulse for both refocusing and coherence transfer. 2D correlation spectroscopy was performed on healthy volunteers in a clinical magnet, at 3 T, within 34 min, for a voxel size of 27 cm(3). This result makes it possible to consider clinical applications. (C) 2002 Elsevier Science B.V. All rights reserved.
The combination of localized 2D H-1 MR correlation spectroscopy and Hadamard encoding allows the simultaneous acquisition of multiple volumes of interest without an increase in the experimental duration, compared to single-voxel acquisition. In the present study, 2D correlation spectra were acquired simultaneously within 20 to 40 min in two voxels located in each hemisphere of the rat brain. An intervoxel distance of 20% of the voxel size was sufficient to limit spatial contamination. The following cerebral metabolites gave detectable crosspeaks : N-acetylaspartate, the glutamate/glutamine pool, aspartate, phosphoethanolamine, glucose, glutathione, taurine, myo-inositols, lactate, threonine, ? -aminobutyric acid, and alanine. Most of the metabolites were measured without contamination of other resonances. (C) 2001 Academic Press.
In this study, skeletal muscle degeneration-regeneration, induced by notexin (a myotoxic substance) injection is studied by Magnetic Resonance Imaging (MRI) and by histological cuts (hematoxylin/eosin and Evans blue colorations). Comparison bem een MR images and histological cuts results (necrosis, myoblasts replication and fusion) permits MR images interpretation. From day 0 to day 13 after notexin injection, injected zone images are very different from those realized in healthy muscles. Preliminary localized 1D H-1-MRS (Magnetic Resonance Spectroscopy) study is also reported. (C) 2001 Academie des sciences/Editions scientifiques et medicales Elsevier SAS.
In the perfused rat heart NMR inversion transfer revealed the existence of a compartment of ATP not exchanging through creatine kinase (CK), as demonstated by an apparent discrepancy between the forward (F-f) and reverse (F-r) CK flux if this compartment was neglected in the analysis [Joubert et al, (2000) Biophys. J. 79, 1-13], To localize this compartment, CK fluxes were measured by inversion of PCr (inv-PCr) or ? ATP (inv-ATP), and the distribution of metabolites between mitochondria and cytosol was studied by subcellular fractionation. Physiological conditions were designed to modify the concentration and distribution of CK metabolites (control, adenylate depletion, inhibition of respiration, KCl arrest). Depending on cardiac activity, mitochondrial ATP (mito-ATP) assessed by fractionation varied from 11% to 30% of total ATP. In addition, the apparent flux discrepancy increased together with mito-ATP (F-f/F-r ranged from 0.85 to 0.50 in inv-PCr and from 1.13 to 1.88 in inv-ATP), Under conditions masking the influence of the ATP-P-i exchange on CK flux, the ATP compartment could be directly quantified by the apparent flux discrepancy ; its size was similar to that of mito-ATP measured by fractionation. Thus NMR inversion technique is a potential tool to assess metabolite compartmentation in the whole organ.
The interpretation of creatine kinase (CK) flux measured by P-31 NMR magnetization transfer in vivo is complex because of the presence of competing reactions, metabolite compartmentation, and CK isozyme localization. In the isovolumic perfused rat heart, we considered the influence of both ATP compartmentation and ATP-P-i exchange on the forward (F-f : PCr —> ATP) and reverse (F-r) CK fluxes derived from complete analysis of inversion transfer. Although F-f should equal F-r because of the steady state, in both protocols when PCr (inv-PCr) or ATP (inv-ATP) was inverted and the contribution of ATP-P-i was masked by saturation of P-i (sat-P-i), F-f/F-r significantly differed from 1 (0.80 +/- 0.06 or 1.32 +/- 0.06, respectively, n = 5). These discrepancies could be explained by a compartment of ATP (f(ATP)) not involved in CK. Consistently, neglecting ATP compartmentation in the analysis of CK in vitro results in an underestimation of F-f/F-r for inv-PCr and its overestimation for inv-ATP. Both protocols gave access to f(ATP) if the system was adequately analyzed. The fraction of ATP not involved in CK reaction in a heart performing medium work amounts to 20-33% of cellular ATP. Finally, the data suggest that the effect of sat-P-i might not result only from the masking of ATP-P-i exchange.
To study the relation among mitochondrial energy supply, cardiac performance, and energy transfer through creatine kinase (CK), two acute models of inhibition of ATP synthesis were compared in the isovolumic acetate-perfused rat heart. Similar impairments of mechanical performance (rate-pressure product, RPP) were achieved by various stepwise decreases in O-2 ; supply (Po-2 down to 20% of control) or by infusing CN (0.15-0.25 mM). The forward CK flux measured by saturation-transfer P-31 NMR spectroscopy was 6.1 +/- 0.4 mM/s in control hearts. Only after severe hypoxia (Po-2
NMR study of membrane biomolecules comes up against a poor solubility in classical solvents. A strategy was elaborated to obtain structural information of peptides in non deuterated phospholipids vesicles. It is based on isotopic (HSQC-NOESY) and homonuclear selective filters, both using a fine water suppression. The method is illustrated with the substance P, a 11-residue membrane neuropeptide.
Muscle degeneration and regeneration were studied by 2D H-1 magnetic resonance spectroscopy (MRS) and histological examination, in an experimental model of muscle injury using a myotoxic snake venom, notexin, The injured muscles produced a very specific MRS signal, corresponding to a tri-unsaturated fatty acid (linolenic acid-like) signal, from day 2 to day 9 after injury, The combination of MRS with histology showed that this signal was associated with a mechanism occurring during myoblast fusion to form myotubes, 2D H-1 MRS is thus a useful non-invasive tool for detecting muscle regeneration in vivo. (C) 1998 Federation of European Biochemical Societies.
Fatty acids are one of the components of living things that nave physiologically significant roles. One group, the phospholipids are a major constituent of cell membranes, while another, the triglycerides act as energy reserves. Nuclear Magnetic Resonance is a non-invasive, atraumatic method that can be used to study these biological materials in living animals. The first part of this article describes the nuclei and experiments (1D and 2D) used in the qualitative and quantitive analysis of mixtures of lipids derivatives. The second part is concerned with studies on lipids in living systems that are placed under specific constraints.
To study the dependence of the forward flux of creatine kinase (CK) on its substrates and products we designed an acute normoxic model of steady-state depletion of phosphocreatine (PCr) and adenylate in the isovolumic acetate-perfused rat heart. Various concentrations of PCr and ATP were induced by prior perfusion with 2 deoxy-D-glucose in the presence of insulin. The apparent rate constant (K-f) and the forward CK flux were measured under metabolic and contractile steady state by progressive saturation-transfer P-31 nuclear magnetic resonance (NMR). At high adenylate content CK flux was constant for a twofold reduction in PCr concentration ([PCr]) ; CK flux was 6.3 +/- 0.6 mM/s (vs. 6.5 +/- 0.2 mM/s in control) because of a doubling of k(f). Although, at the lowest ATP concentration and [PCr], CK flux was reduced by 50%, it nevertheless always remained higher than ATP synthesis estimated by parallel oxygen consumption measurement. NMR-measured flux was compared with the flux computed under the hypothesis of CK equilibrium. CK flux could not be fully predicted by the concentrations of CK metabolites. This is discussed in terms of metabolite and CK isozyme compartmentation.
The proton NMR spectra of regenerating muscle show high resolution fatty acid signals as the spectra of other stressed cells such as ischemic cardiac cells, stimulated immune cells or malignant cells. We report here the in vitro study, by 2D H-1 NMR, of the fusion of myogenic cells. High resolution fatty acid signals are only detected during cell fusion, demonstrating a higher mobility of the acyl chains.
The suitability of two-dimensional (2D) proton spectroscopy for monitoring, in vivo, the changes in levels of brain metabolites induced by cerebral ischemia was investigated in an experimental model of 30-min reversible ischemia induced by four-vessel occlusion in the rat. The resulting data were compared with those obtained by one-dimensional (1D) proton and phosphorus spectroscopy. Phosphorus spectra obtained during ischemia showed significant drops in levels of phosphocreatine (-73%), ß-ATP (-60%), and intracellular pH (to 6.30) and an increase in inorganic phosphate level (905%), 1D and 2D proton spectra showed decreases in the N-acetylaspartate/creatine-phosphocreatine ratio that were not significantly different [-21% (1D) and -32% (2D)]. Similarly, the increases in lactate/creatine-phosphocreatine ratio were not significantly different [2,546% (1D) and 3,020% (2D)].
A new method for analysing complex mixtures, H-1 2D n.m.r., was used to determine polyaromatics in crude gas oil mixtures. 2D NMR overcomes the lack of resolution due to crowded 1D spectra and provides structural information. In particular, TOCSY (total correlation spectroscopy) 2D n.m.r, is well suited to polyaromatics because these molecules give specific 2D fingerprints which can be easily recognized. These patterns were selected and analysed in two ways : (1) specific fingerprint recognition using a pure compound library ; (2) using the mixing time tau(m) of the TOCSY sequence. Variation of tau(m) gives a change in cross-peak intensities. The intensity variation curves are characteristic of spin systems and hence of the structures of compounds. Alkylated substitutions were also studied. The compounds were quantified. This strategy was used to analyse crude gas oils and measure the contents of separated alkylated naphthalene isomers, biphenyls, anthracene, phenanthrene and benzothiophene.
Itaconate biosynthesis was studied in intact cells of high-yield (RC4’) and low-yield (CM85J) strains of the fungus Aspergillus terreus by methods (tracers, nuclear magnetic resonance spectroscopy, and mass spectroscopy) that did not interfere with metabolism, Itaconate formation in RC4’ required de novo protein biosynthesis. Krebs cycle intermediates increased in both strains during the production of itaconic acid. The Embden-Meyerhof-Parnas pathway and the Krebs cycle were shown to be involved in this biosynthesis by using C-14- and C-13-labelled substrates and nuclear magnetic resonance spectroscopy. A metabolic pathway for itaconate formation from glucose in A, terreus is proposed.
MAMMALS react to acute hypoxia with an initial augmentation and a secondary depression of the respiratory rhythm generated by brain stem neuronal networks. To investigate the cytosolic level of energy rich phosphorus metabolites during these responses, we developed P-31 nuclear magnetic resonance spectroscopy of the brain stem. Moderate hypoxia (p(a)O(2) = 40 mmHg, 2 min) caused a reversible 62 +/- 15% respiratory rhythm depression and decreased cytosolic phosphocreatine levels by 43 +/- 11% (p
The conformations of the complexes formed by the ionophore nigericin with Na+ and K+ in CDCl3 solution determined by ROESY NMR and distance geometry computations are in good agreement with the X-ray crystallographic data. The structures with the Na+ ion are more compact than those with the K+ ion, as the latter have a wider cage for the ion.
The cerebral metabolic changes elicited by kainate-induced seizures in the rat were investigated by in vivo combined NMR spectroscopy of P-31 and H-1. Systemic injection of kainate induced no significant changes in cerebral ATP or PCr levels during up to 90 min of continuous, generalised seizures, and the cerebral P-31 spectra showed only a transient mild cerebral acidosis 30 min after kainate administration. In parallel with the changes in intracellular cerebral pH, the H-1 spectra showed a significant increase in lactate, which remained elevated throughout the seizures. These findings indicate that oxidative metabolism does not completely match the increased glycolysis during seizures though the energy homeostasis is maintained. This suggests that oxidative metabolism has a limited capacity to satisfy the brain’s energy needs during the kainate-induced seizures, but that the different pathways of energy production in the brain cells can overcome this limitation. Thus the brain damage associated with this experimental model of epilepsy is not due to extended major failure of the energy supply.
The dynamic effects of the non-competitive NMDA receptor antagonist, MK-801 on brain metabolism were investigated over 105 minutes in unanesthetized rats by proton and phosphorus NMR spectroscopy. MK-801 (0.5 and 5 mg/kg, i.p) induced no changes in intracellular pH, and in phosphocreatine, ATP, and inorganic phosphate levels, indicating that the drug preserved energy and intracellular pH homeostasis. There were transient increases in lactate after both doses of MK-801, suggesting early activation of glycolysis, which was not immediately matched by enhanced oxidative metabolism or by enhanced brood flow. Thereafter, lactate control level was not restored after 0.5 mg/kg whereas it was restored after 5 mg/kg in spite of a sustained metabolic activation.
We have previously demonstrated that 2D H-1 NMR is suitable for studying cerebral metabolism. The same technique was used to study the hind leg muscle of normal (C57BL10) and dystrophic (mdx) mice. The results were compared to preliminary results for cultured muscle cells to determine the origin of fatty acid signals.
Taxol 1 and Taxotere(R) 2 are antitumor compounds interacting with tubulin proteins. In order to find the best conformational fit to the receptor site, the structures of taxotere and twelve analogues showing various in vitro biological activity on tubulin, have been investigated by H-1 NMR spectroscopy and molecular modeling studies. These structures were compared to that of Taxotere(R) 2 obtained by X-ray analysis. The results obtained from these studies suggest that the most active 2R,3’S compounds possess a conformation in which the benzoate group at C-2 holds the side chain in a defined position due Lo hydrophobic interactions between this group and the N-amido or N-carbonyloxy group at C-3’. This situation together with the presence of hydrogen bonding between 2’OH -3’NH and 2’OH-1’C=O gives rise to a specific orientation of the hydroxyl and phenyl groups at C-2’ and C-3’. On the other hand, the 2’S,3’R isomers which display low in vitro biological activity (ie : on tubulin), such as isotaxotere 8, possess a different conformation with no hydrophobic interactions between the side chain and the taxan skeleton.
The synthesis of ortho-ortho’ substituted phenylpyrroles that are susceptible to isomerization of the biaryl type (atropisomerism) was performed using a Michael addition of isocyanoacetates with nitrostyrenes. Atropisomerism in phenylpyrroles 14 and 15 was studied by means of H-1 NMR spectroscopy using chiral lanthanide shift reagents (LSR*). In the case of chiral phenylpyrrole 21, an evaluation of the interconversion parameters between diastereomers (k, ?G(T)*) was attempted.
Homonuclear H-1 2D NMR spectroscopy (COSY experiments at 400 and 600 MHz) were used to study the rat brain in vivo and the rabbit spinal cord and sciatic nerve in vitro. The following metabolites were identified : lactate, alanine, threonine, GABA, glutamine/glutamate, N-acetyl aspartate, aspartate, taurine, inositol derivatives, choline derivatives, and glucose. The sciatic nerve spectra showed characteristic COSY graphs of saturated and unsaturated fatty acids, and linoleic and linolenic type structures were identified.
We have used slice preparation from newborn rats to study the development of the nucleus tractus solitarius neuronal network and brain intracellular phosphorus metabolites. As shown previously on adults, the newborn preparation retains local excitatory and inhibitory synaptic connections and enables study of intrinsic electrical properties in the nucleus tractus solitarius. Electrophysiological investigation of inhibitory synaptic transmission demonstrated a maturationnal step at days 4-6 after birth. Nuclear magnetic resonance spectroscopy of brain slices revealed a metabolic maturation between postnatal days 11 and 17. Results emphasize the differential maturation steps during the postnatal development of rat central nervous system. Possibly, Sudden Infant Death Syndrome may result from the abnormal timing in the occurrence of these steps.
Bioactivity-guided purification of a crude alkaloid extract of Psychotria oleoides has afforded a new alkaloid, psycholeine , together with quadrigemine C , a tetrameric pyrrolidinoindoline compound of unknown stereochemistry. A comparison study of nmr and cd spectra of quadrigemine C and hodgkinsine , a trimeric pyrrolidinoindoline substance, led us to suggest the stereochemistry of quadrigemine C. The structure and configuration of psycholeine was determined by spectroscopic means and chemical correlation with quadrigemine C. Psycholeine interacts with somatostatin receptors and exhibits a somatostatin antagonistic activity on GH secretion by pituitary cells in primary culture.
A concerted mechanism has been demonstrated for the rearrangement of a tetracyclic ion including a bicyclo [4.2.0] octan system to hibaol, using a selective deuteration on the migrating bond. The stereochemistry of the selectively introduced deuterium was determined by three routes : I) comparison of the high field H-1 NMR spectra of the deuterated and undeuterated compounds, using double irradiation ; II) high field H-1 NMR, coupled with molecular mechanics calculations ; III) two dimensional homo and heteronuclear NMR.
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