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Depuis plusieurs milliers d'années, la lumière qu'elle soit naturelle ou artificielle a été utilisée comme traitement de maladies de la peau, des yeux ou contre le cancer. Ce n'est qu'au début du XXe siècle que la science s'est intéressée aux interactions « lumière-vivant ». La compréhension de l'action conjuguée, dénommée « photothérapie dynamique », d'un médicament non toxique à l'obscurité, de l'oxygène et de la lumière a permis de mettre au point un protocole d'éradication de tumeurs bénignes ou malignes et d'anomalies vasculaires. Depuis environ 30 ans, on voit apparaître de nombreuses molécules photoactivables de plus en plus efficaces. Peu de molécules photosensibles ont encore atteint le stade clinique, mais les résultats publiés dans la littérature scientifique montrent que la photothérapie dynamique est en plein développement et a un avenir certain [1-3].

The purpose of this study was to provide proof of concept for a new three-dimensional (3D) radial dynamic contrast enhanced MRI acquisition technique, called "Radial Entire Tumor with Individual Arterial input function dynamic contrast-enhanced MRI" (RETIA dynamic contrast-enhanced MRI), which allows for the simultaneous measurement of an arterial input function in the mouse heart at 2 s temporal resolution and coverage of the whole tumor. Alternating 2D and 3D projections contribute to the 2D heart image or 3D tumor data with a 3-cm field of view. Sixty-four 2D images of the heart are obtained during acquisition of each 3D tumor dataset. In a pilot study, global K(trans) and ve values were measured in four mice, in a respiratory motion-animated subcutaneously implanted breast tumor model. This technique is expected to be most useful for the characterization of microvasculature in motion-animated orthotopic tumors.

Dynamic contrast-enhanced MRI is extensively studied to define and evaluate biomarkers for early assessment of vasculature-targeting therapies. In this study, two-dimensional and three-dimensional radial multi-gradient-echo techniques for dynamic R*2-corrected R1 mapping based on the spoiled gradient recalled signal equation were implemented and validated at 4.7 T. The techniques were evaluated on phantoms and on a respiratory motion animated tumor model. R1 measurements were validated with respect to a standard inversion-recovery spin-echo sequence in a four-compartment phantom covering a range of relaxation rates typically found in tumor tissue. In the range of [0.4, 3] sec−1, R1 differences were less than 10% for both two-dimensional and three-dimensional experiments. A dynamic contrast-enhanced MRI pilot study was performed on a colorectal tumor model subcutaneously implanted in mice at the abdominal level. Low motion sensitivity of radial acquisition allowed image recording without respiratory triggering. Three-dimensional Ktrans maps and significantly different mean Ktrans values were obtained for two contrast agents with different molecular weights. The radial multi-gradient-echo approach should be most useful for preclinical experimental conditions where the tissue of interest experiences physiologic motion, like spontaneous extracerebral tumors developed by transgenic mice, and where dynamic contrast-enhanced MRI is performed with high-relaxivity contrast agents. Magn Reson Med, 2010. © 2010 Wiley-Liss, Inc.

Summary Background Photodynamic therapy is an established cancer treatment in which a photosensitizing agent is activated by exposure to light thus generating cytotoxic reactive oxygen species that cause cellular damage. Methods A new photosensitizer synthesized at Curie Institute was used to treat retinoblastoma xenografts in mice, a glycoconjugated meso substituted porphyrin derivative, that showed some retinoblastoma cell affinity. The longitudinal follow-up of the tumors was carried out by 23Na MRI (without adding exogenous contrast agents) to map the extracellular compartment and to characterize cell packing. Two regimens were followed to target either blood vessels alone or blood vessels and cancer cells simultaneously. Results and conclusions Only the protocol targeting both cancer cells and blood vessels effectively induces cellular death, confirmed by histology at the end of the experiment. Sodium MRI evidences a huge change in the cellular density of tumors only 24 h after a double targeting (vascular and cellular) PDT treatment. We suggest that this change was possibly due to a bystander effect that can be promoted by the intercellular signaling favored by the high cellular density of retinoblastoma. These results indicate that non-invasive 23Na imaging (which detects the tumor response to treatment from very early stages) in association with non-mutagenic therapies represents an effective option for tailored and individualized clinical treatments.

CaVP is a calcium-binding protein from amphioxus. It has a modular composition with two domains, but only the two EF-hand motifs localized in the C-terminal domain are functional. We recently determined the solution structure of this regulatory half (C-CaVP) in the Ca2+-saturated form and characterized the stepwise ion binding. This paper reports the 15N nuclear relaxation rates of the Ca2+-saturated C-CaVP, measured at four different NMR fields (9.39, 11.74, 14.1, and 18.7 T), which were used to map the spectral density function for the majority of the amide HN-N vectors. Fitting the spectral density values at eight frequencies by a model-free approach, we obtained the microdynamic parameters characterizing the global and internal movements of the polypeptide backbone. The two EF-hand motifs, including the ion binding loops, behave like compact structural units with restricted mobility as reflected in the quite uniform order parameter and short internal correlation time (< 20 nsec). Comparative analysis of the two Ca2+ binding sites shows that site III, having a larger affinity for the metal ion, is generally more rigid, and the amide vector in the second residue of each loop is significantly less restricted. The linker fragment is animated simultaneously by a larger amplitude fast motion and a slow conformational exchange on a microsecond to millisecond time scale. The backbone dynamics of C-CaVP characterized here is discussed in relation with other well-characterized Ca2+-binding proteins. Supplemental material: See www.proteinscience.org

The three-dimensional structure of apo-neocarzinostatin (apo-NCS, MW: ca.11000, antitumoral chromophore carrier protein) is based on a seven-stranded antiparallel beta-sandwich, very similar to the immunoglobulin folding domain. We investigated the backbone dynamics of apo-NCS by (13)C-NMR relaxation measurements and molecular dynamics simulation. Model-free parameters determined from the experimental data are compared with a 1.5-nsec molecular simulation of apo-NCS in aqueous solution. This comparison provides an accurate description of both local and collective movements within the protein. This analysis enabled us to correlate dynamic processes with key interactions of this beta-protein. Local motions that could be relevant for the intermolecular association with the ligand are also described.

International audience; Intervertebral disc (IVD) degeneration is a complex process characterized by biochemical and structural changes in both the nucleus pulposus and the anulus fibrosus. In this study, we were able to obtain in vivo magnetic resonance (MR) images of the rabbit spine, with several MR imaging (MRI) contrasts (rho, T(1) and T(2)). We quantified several parameters (T(2), apparent diffusion coefficient, disc height and area) to differentiate between healthy and degenerative IVDs and to characterize the degeneration process. To our knowledge, there has not been any previous in vivo study of rabbit IVDs at high-field MRI (9.4 T). A custom radio frequency (RF) coil for 9.4 T was designed to match rabbit IVD morphology, to study the degeneration in vivo on a model of human lumbar disease. Our new probe, a custom half-birdcage-type coil, obtains the necessary exploration depth while meeting the requirements for signal homogeneity and sensitivity of the study. This design addresses some of the difficulties with constructing RF coils at high field strengths.

Sodium is the second best nucleus in living systems MRI after proton, but it is the first one concerning its sensitivity to the electrical configuration of the environment, due to its quadrupolar relaxation properties. Retrieving accurate relaxation data from low signal-to-noise ratio (SNR) experiments implies using post-processing procedures in order to fit correctly the decaying relaxation curves. The conventional image reconstruction method is introducing a positive bias due to the rician noise contribution. Phasing the images restores its initial Gaussian characteristic. Linear prediction (LP) forward of the decays improves the SNR of the last points and calculates missing points. An objective fitting method, based on singular value decomposition (SVD) allows us to determine the number of exponentials existing in the decay and its accurate fitting parameters. Sodium ions' spin–spin relaxation holds great potential to become an endogeneous marker for tumor diagnosis and for therapy response, since different regions of a mouse tumoral liver bear distinguishable relaxation parameters as compared to control.

A double-delay SR-MGE-SNAP sequence allowing simultaneous T1 and T2* measurement was developed for integrating arterial input function (AIF) measurement into DCE MRI. Implemented on a 4.7-T animal MR system, this technique was applied to mice with colorectal tumor xenografts. AIF, measured in the mouse heart, was modeled by a bi-exponential function, whereas tumor K(trans) and v(e) parameter maps were obtained from analysis with a two- compartment model using an individually measured AIF. AIF analysis of T2*-corrected data yielded A1 = 9.2 +/- 4.3 kg/l, A(2) = 4.2 +/- 0.8 kg/l, m1 = 2.3 +/- 1.1 min(-1), and m2 = 0.05 +/- 0.02 min(-1). The mean initial plasma concentration C ( p )(t = 0) = 8.0 +/- 2.7 mM was compatible with estimated 8.6 mM. Without T2*-correction distribution phase parameters A1, m1, and C(p)(t = 0) were underestimated. In tumors, neglect of T2* effects yielded mean K(trans) values which were reduced by 14% (P < 0.05), whereas v(e) showed only a slight non-significant reduction. Simultaneous measurement of DeltaR1 and DeltaR2* studied in highly and poorly vascularized and (pre-)necrotic tumor regions revealed complementary behavior of both parameters with respect to vascular properties. In conclusion, the presented measurement technique is a promising tool for dynamic MRI applications studied in animal models at high field strengths and/or with CA of high relaxivities, as it combines classical DCE MRI integrating AIF assessment with dynamic T2* measurement.

Renal function is highly correlated with the sodium concentration gradient along the corticomedullary axis. The application of 3D high-resolution sodium magnetic resonance imaging (MRI) provided a means to quantify in vivo the spatial and temporal changes in renal tissue sodium concentration under normal and diuretic conditions. A detailed, pixel-by-pixel analysis of the intact rat kidney sodium MR images yielded a quantitative measure of the corticomedullary sodium gradient before and at early and later times after the administration of two distinct diuretic agents, furosemide and mannitol. Furosemide, a loop diuretic, induced a fivefold reduction in the cortical-outer medullary sodium gradient, whereas mannitol, an osmotic diuretic, did not affect this gradient. Both diuretics induced a 50% decrease in the sodium concentration of the inner medulla; however, mannitol exerted its effect twice as fast as furosemide with a 2.5-min exponential decay constant. These specific changes were attributed to the different mechanism of action and site of activity of each diuretic agent. Thus, high-resolution (23)Na MRI offers a unique, noninvasive tool for functional imaging of the kidney physiology.

We are presenting, in this paper, a simple, easily reproducible and easily tuneable design for home-built birdcage probes inducing a linearly polarised B1 field. A simple tuning method is proposed which preserves the induced field homogeneity that characterises in general the birdcage design. This is achieved by adding two extra rings (link rings) that allow one to connect two opposed points on the birdcage end rings via tuning capacitors. All the development stages, from design to practical realisation, are presented, step by step, in order to facilitate experimentalist work. Some images obtained with the birdcage probe are presented, accompanied by the B1 maps in transverse as well as longitudinal planes. Finally, a few suggestions are made for obtaining a circularly polarised magnetic field configuration.

Noninvasive nuclear magnetic resonance was used to measure the relaxation decay curves of naturally occurring 23Na ions in several biological systems. Experimental results showed an increase of membrane bound population for pathologic samples as compared with control. The bound sodium population was put in evidence using singular value decomposition method. Thus, the singular values that are obtained without any a priori from the fitting the relaxation decay curves are a new parameter in characterizing the cellular state. In the presence of artificial biological membranes, 23Na bound strongly to membranes containing phosphatidylcholine (PC) and phosphatidylserine (PS), but not to membranes consisting of only PC. A large bound population also appeared in the presence of apoptotic epithelial cells, which are known to translocate PS to the cell surface. A role for PS was confirmed by showing that sodium binds to the surface of epithelial cells infected with Chlamydia psittaci, and the amplitude of the bound population increases with a time-course similar to the appearance of PS on the surface of dying cells. Finally, this approach could distinguish between normal perfused liver and liver undergoing ischemia, due most likely to the exposure of surface PS on apoptotic and necrotic cells in the damaged tissue. Taken together, these studies demonstrate that the analysis of 23Na relaxation decay curves could reveal the presence of cells undergoing apoptosis and/or necrosis in living tissues. Noninvasive 23Na NMR measurements could thus be envisioned for controlling the quality of organs before transplantation, for the detection of asymptomatic infections that result in death of the host cell or inflammation of the tissue, and for characterizing the efficiency of novel apoptosis-inducing drugs to treat cancer.

Imaging of the vasculature and its functioning over the entire lesion may significantly aid in cancer diagnosis, assessment of prognosis, and therapeutic evaluation. In the current study we present a dynamic three-dimensional deuterium magnetic resonance imaging method that determines the intravascular volume fraction and water perfusion rate at a resolution of 2 mm(2)/pixel. The method was tested and utilized to characterize the vasculature of orthotopic MCF7 human breast cancer tumors in CD1-NU athymic mice. A new algorithm based on Patlak's kinetic model was developed to analyze the dynamic images acquired during and after termination of infusion with deuterated water. The resulting parametric maps spanned a wide range from 0.4 to 35.2% for the intravascular volume fraction and from 4 x 10(-6) to 3.9 x 10(-3) min(-1) for the perfusion rate and exhibited high intratumoral and intertumoral heterogeneity at both parameters. The intravascular volume fraction did not correlate with the corresponding perfusion rate, demonstrating the irregular outgrowth of tumor neovascularization. Averaging the data or analyzing at spatially degraded resolution completely masked the presence of both "hot spots" and hypoxic loci, highlighting the critical importance of high spatial resolution. The method is applicable to other types of tumors and animal models and may be extended to humans.

Summary Background PDT represents a very localized and non-mutagen antitumoral treatment using a photosensitive molecule (porphyrin family) light activated. The first way of cell damage is a direct one, active on the very site where ROSs have been produced. The second one is indirect by activating and transmitting the processes of cellular death signaling. In order to seek for a better characterization of the photo-biology involved in in vivo PDT and to better understand the differences on the treatment outcome, we have used three different human retinoblastomas xenografted on mice. Methods Mice were treated according to the double targeting protocol exposed in a previous paper. One i.v. dose (0.6 mg/kg) of PS was followed by a second dose, separated by a 3 h interval (double targeting PDT). As a consequence both cancer cells and blood vessels were targeted. The treatment was repeated two times, at 4 days interval. Results First of all, sodium MRI revealed qualitative differences in the sodium average content of the three retinoblastoma lines before treatment. After the PDT treatments the tumor responses were different between the lines as revealed by sodium MRI and later on by histology. Conclusions We have put into evidence that PDT is accompanied by a bystander effect that may propagate the cellular death triggered by the initial photoreaction. This effect is highly dependent on the cellular density of the tissue; therefore this factor is to be taken into account in clinical PDT protocols.

The N-Boc O-tert-butyldimethysilyl-substituted hexa-β-peptide methyl ester 18 was constructed from the O-TBS ether of (−)-(2R, 3S)-phenylisoserine. By NMR, it was determined that this homo β-peptide adopts a highly stable β-strand-type secondary structure in chloroform solution, which is stabilized by both hydrophobic interactions involving the OTBS methyl groups of residues i and i + 2, and inter-(five-membered)/intra (six-membered)-residue H-bonding interactions. These interactions are systematically repeated along the peptide chain and, thereby, operate in concert to stabilize the observed conformation of 18.

In the study of protein dynamics by (13)C or (15)N relaxation measurements different models from the Lipari-Szabo formalism are used in order to determine the motion parameters. The global rotational correlation time tau(R) of the molecule must be estimated prior to the analysis. In this Communication, the authors propose a new approach in determining an accurate value for tau(R) in order to realize the best fit of R(2) for the whole sequence of the protein, regardless of the different type of motions atoms may experience. The method first determines the highly structured regions of the sequence. For each corresponding site, the Lipari-Szabo parameters are calculated for R(1) and NOE, using an arbitrary value for tau(R). The chi(2) for R(2), summed over the selected sites, shows a clear minimum, as a function of tau(R). This minimum is used to better estimate a proper value for tau(R).

Two related oncogenes, TCL1 and MTCP1, are overexpressed in certain T-cell prolymphocytic leukemias as a result of chromosomal rearrangements that involve the translocation of one T-cell receptor gene to either chromosome 14q32 or Xq28, respectively. The human oncoprotein p13 MTCP1 is coded by the MTCP1 gene and its primary sequence is highly and only homologous to that of p14 TCL1 , the product of TCL1. These two proteins likely represent the first members of a new family of oncogenic proteins. A previous model of the three-dimensional solution structure of p13 MTCP1 was determined recently using exclusively homonuclear proton two-dimensional NMR methods and, almost simultaneously, high-resolution crystal structures of p13 MTCP1 and p14 TCL1 appeared in the literature. In order to gain more insight into the details of the solution structure, we uniformly labeled p13 MTCP1 with nitrogen-15. The refined structure benefits from 520 additional NOEs, extracted from either 15N-edited 3D experiments or homonuclear 2D NOESY recorded at 800 MHz, and from a nearly complete set of φ angular restraints. Measurements of 15N spin relaxation times and heteronuclear 15N{1H}NOEs at two magnetic field strengths provided additional insights into the dynamics of the protein backbone. On the basis of these new results, a putative binding surface for this particular class of oncogenes is discussed.

MTCP1 (for Mature-T-Cell Proliferation) was the first gene unequivocally identified in the group of uncommon leukemias with a mature phenotype. The three-dimensional solution structure of the human p8 MTCP1 protein encoded by the MTCP1 oncogene has been previously determined by homonuclear proton two-dimensional NMR methods at 600 MHz: it consists of an original scaffold comprising three a-helices, associated with a new cysteine motif. Two of the helices are covalently paired by two disulfide bridges, forming an a-hairpin which resembles an antiparallel coiled-coil. The third helix is orientated roughly parallel to the plane defined by the a-antiparallel motif and appears less well defined. In order to gain more insight into the details of this new scaffold, we uniformly labeled with nitrogen-15 a mutant of this protein (C12A-p8 MTCP1 )i n which the unbound cysteine at position 12 has been replaced by an alanine residue, thus allowing reproducibly high yields of recombinant protein. The refined structure benefits from 211 additional NOEs, extracted from 15 N-edited 3D experiments, and from a nearly complete set of # angular restraints allowing the estimation of the helical content of the structured part of the protein. Moreover, measurements of 15 N spin relaxation times and heteronuclear 15 N{ 1 H}NOEs provided additional insights into the dynamics of the protein backbone. The analysis of the linear correlation between J(0) and J(&) was used to interpret relaxation parameters. It appears that the apparent relative disorder seen in helix III is not simply due to a lack of experimental constraints, but associated with substantial contributions of sub-nanosecond motions in this segment. Abbreviations: 2D (3D), two-dimensional (three-dimensional); NOE, nuclear Overhauser enhancement; 15 N{ 1 H}, heteronuclear 15 N nuclear Overhauser enhancement; NOESY, nuclear Overhauser enhancement spectroscopy; DQF-COSY, double-quantum-filtered scalar-correlated spectroscopy; z-TOCSY, z-filtered total correlation spectroscopy; HSQC, heteronuclear single quantum correlation; HMQC, heteronuclear multiple quantum correlation; RN(Nz )( R 1), heteronuclear 15 N longitudinal relaxation rate; R N(Nxy )( R 2), heteronuclear 15 N transverse relaxation rate; RN(Hz!Nz), cross-relaxation rate between 15 N and its attached amide proton; rmsd, root mean square deviation; PCR, polymerase chain reaction; DTT

An artificial hemoglobin-like domain has been constructed by engineering the gene coding for the multi-domain flavohemoprotein from the bacterium Erwinia chrysanthemi. This domain was designed by molecular modelling, cloned and over-expressed in Escherichia coli. The holo-protein was obtained in large quantities after extraction from inclusion bodies and refolding in presence of alkaline hemin. The purified 140-residue domain was studied and characterized to gain new insights into the biochemical function of the recombinant domain and the biological role of this new flavohemoprotein. The structural and functional features of this domain in solution were studied using far-ultraviolet circular dichroism, resonance Raman, proton-NMR spectroscopy, flash laser photolysis and molecular modelling. The recombinant domain is shown to be folded properly and active. This hemoglobin-like domain is able to bind oxygen and carbon monoxide with very high affinity. It exhibits a rapid auto-oxidation which may explain its tight association with a flavin containing reductase domain. A functional model of this hemoglobin is discussed and compared with the X-ray structures of other hemoproteins.

The Chlamydia species are obligate intracellular bacteria that proliferate only within the infected cell. Since the extracellular bacteria are metabolically inert and there are no cell-free systems for characterizing Chlamydia metabolism, we studied metabolic changes related to ATP synthesis and glycolysis in HeLa cells infected with Chlamydia psittaci during the course of the 2-day infection cycle using noninvasive 31P and 13C NMR methods. We find that the infection stimulates ATP synthesis in the infected cell, with a peak of ATP levels occurring midway through the infection cycle, when most of the metabolically active bacteria are proliferating. The infection also stimulates synthesis of glutamate with a similar time course as for ATP. The stimulation is apparently due to an enhancement in glucose consumption by the infected cell, which also results in an increased rate of lactate production and glutamate synthesis as well as higher glycogen accumulation during the infection. Concurrently, infection leads to an increase in the expression of the glucose transporter, GLUT-1, on HeLa cells, which may account for the enhanced glucose consumption. The chlamydiae are thus able to stimulate glucose transport in the host cell sufficiently to compensate for the extra energy load on the cell represented by the infection.

International audience; 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.

The advantages of band-filtering schemes, based on the selection of an inversion profile, over selective 90° excitation pulse are presented. The performances of two “user-friendly” methods based on such a scheme, DANTE-Z and Spin Pinging, are evaluated. As expected, the two methods give similar results, but, among other things, DANTE-Z is easier to implement on a “routine” spectrometer than Spin Pinging and is less sensitive to relaxation phenomena, especially those related toT2. Moreover, the advantages of using high-power pulse-width-modulated DANTE trains instead of low-power “soft” pulse are emphasized. In association with DANTE-Z or Spin Pinging, this alternative avoids the tedious calibration of BURP pulses and amounts to the calibration of a conventional π/2 “hard” pulse.

FKBP59, a 59-kDa FK506 binding protein, was discovered in heterooligomeric complexes containing nontransformed, non-DNA binding, steroid receptors. Sequence similarity search and secondary structure prediction suggested that the protein has a multi-domain organization, the N-terminal domain having a great similarity to human FKBP12 (12-kDa FK506-binding protein). FKBP59 binds immunosuppressant FK506 and has peptidylprolyl cis-trans-isomerase activity, both properties being localized in the N-terminal domain (FKBP59-I). In order to characterize its conformational features and to better understand its biological significance, we overexpressed and 15N-labeled this domain (149 amino acids) in Escherichia coli and initiated an NMR structural study in solution. Almost complete sequence-specific assignment of the 1H and 15N resonances was achieved using two-dimensional and three-dimensional homonuclear and heteronuclear experiments. Localization of the secondary structure elements was derived essentially from C alpha H chemical shift distribution along the sequence, the short-range and medium-range NOE connectivities and exchange kinetics of amide protons. The domain has a structured part comprising six beta-strands and a three-turn alpha-helix between K87 and M96. The first 17 residues are highly flexible and show no regular secondary structure. The beta-sheet structure, derived from long-range connectivities between backbone protons, consists of six beta-strands defined as follows: B1, V22-I24; B2, V32-K37; B3, D50-L61; B4, T64-S68 and F76-L80; B5, E100-K107; B6, L127-F137. They are organized in an antiparallel beta-sheet with the connecting topology +1, +3, +1, -3, +1. The alpha-helix connects strand B4 to strand B5. Globally, the structure of FKBP59-I, derived from the present work, is similar to the NMR-derived structures of uncomplexed FKBP12. However, several conformational differences were noted at this level of structural analysis. The beta-sheet of the FKBP59 domain has an additional strand at the N-terminal and the alpha-helix is longer by about one helical turn. In addition, strand B4 has two components, separated by a large bulge (seven residues); the first component was observed in the X-ray or NMR structures of complexed FKBP12 but not in the NMR-derived, uncomplexed structure.

The purpose of this study was to provide proof of concept for a new three-dimensional (3D) radial dynamic contrast enhanced MRI acquisition technique, called "Radial Entire Tumor with Individual Arterial input function dynamic contrast-enhanced MRI" (RETIA dynamic contrast-enhanced MRI), which allows for the simultaneous measurement of an arterial input function in the mouse heart at 2 s temporal resolution and coverage of the whole tumor. Alternating 2D and 3D projections contribute to the 2D heart image or 3D tumor data with a 3-cm field of view. Sixty-four 2D images of the heart are obtained during acquisition of each 3D tumor dataset. In a pilot study, global K(trans) and ve values were measured in four mice, in a respiratory motion-animated subcutaneously implanted breast tumor model. This technique is expected to be most useful for the characterization of microvasculature in motion-animated orthotopic tumors.

Nearly complete assignment of the protonated carbon resonances of apo-neocarzinostatin, a 113-amino acid antitumor antibiotic carrier protein, has been achieved at natural 13C abundance using heteronuclear 2D experiments. Most of the cross peaks in the proton-carbon correlation map were identified by the combined use of HMQC, HMQC-RELAY and HMQC-NOESY spectra, using already published proton chemical shifts. However, double-DEPT and triple-quantum experiments had to be performed for the edition of CH and CH2 side-chain groups, respectively, which were hardly visible on HMQC-type maps. The triple-quantum pulse sequence was adapted from its original scheme to be applicable to a natural abundance sample. The correlation between carbon chemical shifts and the apo-neocarzinostatin structure is discussed. In particular, 13C alpha secondary shifts correlate well with the backbone conformation. These shifts also yield information about the main-chain flexibility of the protein. Assignments reported herein will be used further for interpretation of carbon relaxation times in a study of the internal dynamics of apo-neocarzinostatin.

Summary Background Previous in vivo studies on photodynamic therapy (PDT)-treated, high cellular density tumors showed evidences of a bystander effect accompanying the therapy, cellular death continuing beyond the limits of the photochemical reactions in time and space. This process is generated by the initially damaged cells on the light pathway. The aim of this study was to determine if the bystander effect may be induced as well in colorectal xenografted tumors (less compact structure) and if the cellular signaling depends primarily on cellular proximity or not. Methods The photosensitizer was a glycoconjugated, meso substituted porphyrin derivative synthesized at Institut Curie. The longitudinal follow-up of the tumors was carried out by 23Na/1H MRI, ideal imaging modality for mapping the extracellular compartment. Two regimens were followed in order to target either blood vessels alone or blood vessels and cancer cells simultaneously. Results The antivascular PDT did not succeed to arrest the tumors growth at the end of the follow-up. For double targeting PDT, we managed to stop the tumoral evolution. Sodium MRI evidenced a bystander effect. Conclusion The results obtained showed that the bystander effect is more difficult to induce for the type of colorectal tumors used in this work. It needs a double treatment, 4 days apart, in order to be promoted.

Dynamic contrast-enhanced MRI is extensively studied to define and evaluate biomarkers for early assessment of vasculature-targeting therapies. In this study, two-dimensional and three-dimensional radial multi-gradient-echo techniques for dynamic R*(2)-corrected R(1) mapping based on the spoiled gradient recalled signal equation were implemented and validated at 4.7 T. The techniques were evaluated on phantoms and on a respiratory motion animated tumor model. R(1) measurements were validated with respect to a standard inversion-recovery spin-echo sequence in a four-compartment phantom covering a range of relaxation rates typically found in tumor tissue. In the range of [0.4, 3] sec(-1), R(1) differences were less than 10% for both two-dimensional and three-dimensional experiments. A dynamic contrast-enhanced MRI pilot study was performed on a colorectal tumor model subcutaneously implanted in mice at the abdominal level. Low motion sensitivity of radial acquisition allowed image recording without respiratory triggering. Three-dimensional K(trans) maps and significantly different mean K(trans) values were obtained for two contrast agents with different molecular weights. The radial multi-gradient-echo approach should be most useful for preclinical experimental conditions where the tissue of interest experiences physiologic motion, like spontaneous extracerebral tumors developed by transgenic mice, and where dynamic contrast-enhanced MRI is performed with high-relaxivity contrast agents.

Photodynamic therapy (PDT) is a recent approach for the treatment of small cancerous tumours, on-surface or accessible by endoscopy in which a dye (usually a tetrapyrrolic macrocycle) absorbs light and generates cytotoxic reactive oxygen species leading to cellular damage. Retinoblastoma (Rb) is a rare intraocular tumour of childhood. All the multifocal forms are hereditary and constitute a syndrome of genetic predisposition in the cancer. The current treatments with etoposide or carboplatine expose the patient to the late risk of second cancer. The use of PDT as cancer therapy is particularly attractive due to the use of few mutagenic and non-toxic photosensitizers (PS) prior light excitation and to the localized tumour illumination. The photoefficiency towards Rb of a glycoconjugated porphyrin is discussed and compared with the results obtained with a second-generation photosensitizer, the Foscan. Some in vivo results on an animal model of Rb are presented by a point of view of photoefficiency, biodistribution, pharmacokinetic and longitudinal follow-up of the PDT effect using a new non-invasive method of magnetic resonance imaging of real-time. Photodynamic treatments in association with non-invasive sodium imaging open ways for new treatment tailoring or treatment individualization of retinoblastoma in clinic.

A peptide of 17 amino acid residues Ac-L-K-W-K-K-L-L-K-L-L-K-K-L-L-K-L-G-NH2, designed to form an amphiphilic basic alpha-helix [DeGrado, W.F., Prendergast, F. G., Wolfe, H. R., Jr., & Cox, J. A. (1985) J. Cell. Biochem. 29, 83-93], was labeled with 15N at positions 1, 7, 9, and 10. Homo- and heteronuclear NMR techniques were used to characterize the conformational changes of the peptide when it binds to calmodulin in the presence of Ca2+ ions. The spectrum of the free peptide in aqueous solution at pH 6.3 and 298 K was completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide populates, to a significant extent, an alpha-helix conformational state, in agreement with circular dichroism measurements under similar physicochemical conditions. 15N-edited 1D spectra and 15N(omega 2)-half-filtered two-dimensional NMR experiments on the peptide in a 1:1 complex with calmodulin allowed assignment of half of the amide proton resonances and three C alpha H resonances of the bound peptide. The observed NOE connectivities between the peptide backbone protons are indicative of a stable helical secondary structure spanning at least the fragment L1-K11. The equilibrium and dynamic NMR parameters of the bound peptide are discussed in terms of a molecular interaction model.

The metabolic effects of R-phenylisopropyladenosine ( R-PIA), an agonist of adenosine A, receptors, were studied by in vivo 31P NMR spectroscopy before, during, and after 30 min of reversible forebrain ischemia in the rat. R-PIA had no effect on cerebral metabolism before ischemia. During a 30-min ischemia, R-PIA reduced the decrease in phosphocreatine (43 ± 11% of the control level at the end of ischemia vs. 27 ± 9% in the reference group) and ATP (58 ± 12% vs. 40 ± 23%) and the increase in inorganic phosphate (672 ± 210% vs. 905 ± 229%). The intracellular acidosis elicited by ischemia was also less in the treated group (pH of 6.40 ± 0.10 vs. 6.30 ± 0.10). Recirculation was associated with a faster recovery of PCr, ATP, Pi, and pHi to control levels in the treated group than in the reference group. It is concluded that adenosine protects against ischemic injury by mechanisms that include metabolic protection.

The metabolic effects of kynurenate, an endogenous excitatory amino acid antagonist, were studied by in vivo 31P-NMR spectroscopy before, during and after reversible forebrain ischemia in the rat. Kynurenate had no effect on cerebral metabolism before ischemia. During a 30-min ischemia, kynurenate protected against the decrease in phosphocreatine (up to -55 +/- 3% vs -73 +/- 3% in the reference group) and the increase in inorganic phosphate (up to +479 +/- 39% vs +805 +/- 66%), whereas there was no statistical difference in the decrease in intracellular pH (up to 6.37 +/- 0.05 vs 6.30 +/- 0.03) and ATP (up to -60 +/- 3% vs -60 +/- 7%). The recovery of PCr, Pi, and pHi to control levels during recirculation was faster in the treated group than in the reference group, whereas the time course of ATP recovery was similar in both groups. We conclude that kynurenate protects against neuronal loss, as previously reported, by mechanisms other than metabolic protection.

The sequence-specific resonance assignment of apo-neocarzinostatin from Streptomyces carzinostaticus was carried out from two-dimensional proton-NMR spectra. The assignments were obtained for the backbone protons of 111 of the 113 residues of the protein, missing the two C alpha H of one glycine but including 3 of the 4 prolines. The majority of side chain protons were also assigned. The secondary structure derived from the analysis of sequential connections corresponds to ten beta-strands separated by clearly identified loops and turns. Inter-strand connectivities and slowly exchanging amide protons confirm the presence of the two disulfide bridges from Cys37 to Cys47 and from Cys88 to Cys93 and indicate a global folding similar to that of the similar proteins, actinoxanthin and macromomycin, for which crystallographic data are available.

Hemoglobin Saint Mandé (beta N102Y) is a low-affinity mutant with the substitution site situated in the quaternary-sensitive alpha 1 beta 2 interface. In adult hemoglobin the Asn102 beta contributes to the stability of the liganded (R) state, forming a hydrogen bond with Asp94 alpha. The quaternary and tertiary perturbations subsequent to the Tyr for Asn substitution in monocarboxylated hemoglobin Saint Mandé have been investigated by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis of the one-dimensional NMR spectra of the liganded and unliganded samples in 1H2O provides evidence that both R and T quaternary structures of Hb Saint Mandé are different from the corresponding ones in HbA. In the monocarboxylated form of the mutant hemoglobin, at acid pH, we have observed the disappearance of an R-type hydrogen bond and the appearance of a new one whose proton resonates like a deoxy T marker. Using two-dimensional NMR methods and on the basis of previous results on the monocarboxylated HbA, we have obtained a significant number of resonance assignments in the spectra of monocarboxylated Hb Saint Mandé at pH 5.6 in the presence or absence of a strong allosteric effector, inositol hexaphosphate. This enabled us to characterize the tertiary conformational changes (relative to the liganded normal hemoglobin) triggered by the quaternary-state modification. The observed structural variations are confined within the heme pocket regions but concern both the alpha and beta subunits. Most of them, localized in the C, F, G, and FG segments, could result directly from the side-chain substitution, while others, such as Leu141 beta, could be explained only by long-range interactions.