Different internalization properties of the alpha1a- and alpha1b-adrenergic receptor subtypes: the potential role of receptor interaction with beta-arrestins and AP50.

The internalization properties of the alpha1a- and alpha1b-adrenergic receptors (ARs) subtypes transiently expressed in human embryonic kidney (HEK) 293 cells were compared using biotinylation experiments and confocal microscopy. Whereas the alpha1b-AR displayed robust agonist-induced endocytosis, the alpha1a-AR did not. Constitutive internalization of the alpha1a-AR was negligible, whereas the alpha1b-AR displayed significant constitutive internalization and recycling. We investigated the interaction of the alpha1-AR subtypes with beta-arrestins 1 and 2 as well as with the AP50 subunit of the clathrin adaptor complex AP2. The results from both coimmunoprecipitation experiments and beta-arrestin translocation assays indicated that the agonistinduced interaction of the alpha1a-AR with beta-arrestins was much weaker than that of the alpha1b-AR. In addition, the alpha1a-AR did not bind AP50. The alpha1b-AR mutant M8, lacking the main phosphorylation sites in the receptor C tail, was unable to undergo endocytosis and was profoundly impaired in binding beta-arrestins despite its binding to AP50. In contrast, the alpha1b-AR mutant DeltaR8, lacking AP50 binding, bound beta-arrestins efficiently, and displayed delayed endocytosis. RNA interference showed that beta-arrestin 2 plays a prominent role in alpha1b-AR endocytosis. The findings of this study demonstrate differences in internalization between the alpha1a- and alpha1b-AR and provide evidence that the lack of significant endocytosis of the alpha1a-AR is linked to its poor interaction with beta-arrestins as well as with AP50. We also provide evidence that the integrity of the phosphorylation sites in the C tail of the alpha1b-AR is important for receptor/beta-arrestin interaction and that this interaction is the main event triggering receptor internalization.

Development, optimization, and validation of novel anti-TEM1/CD248 affinity agent for optical imaging in cancer.

Tumor Endothelial Marker-1 (TEM1/CD248) is a tumor vascular marker with high therapeutic and diagnostic potentials. Immuno-imaging with TEM1-specific antibodies can help to detect cancerous lesions, monitor tumor responses, and select patients that are most likely to benefit from TEM1-targeted therapies. In particular, near infrared(NIR) optical imaging with biomarker-specific antibodies can provide real-time, tomographic information without exposing the subjects to radioactivity. To maximize the theranostic potential of TEM1, we developed a panel of all human, multivalent Fc-fusion proteins based on a previously identified single chain antibody (scFv78) that recognizes both human and mouse TEM1. By characterizing avidity, stability, and pharmacokinectics, we identified one fusion protein, 78Fc, with desirable characteristics for immuno-imaging applications. The biodistribution of radiolabeled 78Fc showed that this antibody had minimal binding to normal organs, which have low expression of TEM1. Next, we developed a 78Fc-based tracer and tested its performance in different TEM1-expressing mouse models. The NIR imaging and tomography results suggest that the 78Fc-NIR tracer performs well in distinguishing mouse- or human-TEM1 expressing tumor grafts from normal organs and control grafts in vivo. From these results we conclude that further development and optimization of 78Fc as a TEM1-targeted imaging agent for use in clinical settings is warranted.

The TPTE gene family: cellular expression, subcellular localization and alternative splicing.

The human TPTE (Transmembrane Phosphatase with TEnsin homology) gene family encodes a PTEN-related tyrosine phosphatase with four potential transmembrane domains. Chromosomal mapping revealed multiple copies of the TPTE gene on chromosomes 13, 15, 21, 22 and Y. Human chromosomes 13 and 21 copies encode two functional proteins, TPIP (TPTE and PTEN homologous Inositol lipid Phosphatase) and TPTE, respectively, whereas only one copy of the gene exists in the mouse genome. In the present study, we show that TPTE and TPIP proteins are expressed in secondary spermatocytes and/or prespermatids. In addition, we report the existence of several novel alternatively spliced isoforms of these two proteins with variable number of transmembrane domains. The latter has no influence on the subcellular localization of these different peptides as shown by co-immunofluorescence experiments. Finally, we identify another expressed TPTE copy, mapping to human chromosome 22, whose transcription appears to be under the control of the LTR of human endogenous retrovirus RTVL-H3.

Profile order and time-dependent artifacts in contrast-enhanced coronary MR angiography at 3T: origin and prevention.

To enhance the clinical value of coronary magnetic resonance angiography (MRA), high-relaxivity contrast agents have recently been used at 3T. Here we examine a uniform bilateral shadowing artifact observed along the coronary arteries in MRA images collected using such a contrast agent. Simulations were performed to characterize this artifact, including its origin, to determine how best to mitigate this effect, and to optimize a data acquisition/injection scheme. An intraluminal contrast agent concentration model was used to simulate various acquisition strategies with two profile orders for a slow-infusion of a high-relaxivity contrast agent. Filtering effects from temporally variable weighting in k-space are prominent when a centric, radial (CR) profile order is applied during contrast infusion, resulting in decreased signal enhancement and underestimation of vessel width, while both pre- and postinfusion steady-state acquisitions result in overestimation of the vessel width. Acquisition during the brief postinfusion steady-state produces the greatest signal enhancement and minimizes k-space filtering artifacts.

Source wavelet estimation for waveform inversion of crosshole georadar data

AbstractFor a wide range of environmental, hydrological, and engineering applications there is a fast growing need for high-resolution imaging. In this context, waveform tomographic imaging of crosshole georadar data is a powerful method able to provide images of pertinent electrical properties in near-surface environments with unprecedented spatial resolution. In contrast, conventional ray-based tomographic methods, which consider only a very limited part of the recorded signal (first-arrival traveltimes and maximum first-cycle amplitudes), suffer from inherent limitations in resolution and may prove to be inadequate in complex environments. For a typical crosshole georadar survey the potential improvement in resolution when using waveform-based approaches instead of ray-based approaches is in the range of one order-of- magnitude. Moreover, the spatial resolution of waveform-based inversions is comparable to that of common logging methods. While in exploration seismology waveform tomographic imaging has become well established over the past two decades, it is comparably still underdeveloped in the georadar domain despite corresponding needs. Recently, different groups have presented finite-difference time-domain waveform inversion schemes for crosshole georadar data, which are adaptations and extensions of Tarantola's seminal nonlinear generalized least-squares approach developed for the seismic case. First applications of these new crosshole georadar waveform inversion schemes on synthetic and field data have shown promising results. However, there is little known about the limits and performance of such schemes in complex environments. To this end, the general motivation of my thesis is the evaluation of the robustness and limitations of waveform inversion algorithms for crosshole georadar data in order to apply such schemes to a wide range of real world problems.One crucial issue to making applicable and effective any waveform scheme to real-world crosshole georadar problems is the accurate estimation of the source wavelet, which is unknown in reality. Waveform inversion schemes for crosshole georadar data require forward simulations of the wavefield in order to iteratively solve the inverse problem. Therefore, accurate knowledge of the source wavelet is critically important for successful application of such schemes. Relatively small differences in the estimated source wavelet shape can lead to large differences in the resulting tomograms. In the first part of my thesis, I explore the viability and robustness of a relatively simple iterative deconvolution technique that incorporates the estimation of the source wavelet into the waveform inversion procedure rather than adding additional model parameters into the inversion problem. Extensive tests indicate that this source wavelet estimation technique is simple yet effective, and is able to provide remarkably accurate and robust estimates of the source wavelet in the presence of strong heterogeneity in both the dielectric permittivity and electrical conductivity as well as significant ambient noise in the recorded data. Furthermore, our tests also indicate that the approach is insensitive to the phase characteristics of the starting wavelet, which is not the case when directly incorporating the wavelet estimation into the inverse problem.Another critical issue with crosshole georadar waveform inversion schemes which clearly needs to be investigated is the consequence of the common assumption of frequency- independent electromagnetic constitutive parameters. This is crucial since in reality, these parameters are known to be frequency-dependent and complex and thus recorded georadar data may show significant dispersive behaviour. In particular, in the presence of water, there is a wide body of evidence showing that the dielectric permittivity can be significantly frequency dependent over the GPR frequency range, due to a variety of relaxation processes. The second part of my thesis is therefore dedicated to the evaluation of the reconstruction limits of a non-dispersive crosshole georadar waveform inversion scheme in the presence of varying degrees of dielectric dispersion. I show that the inversion algorithm, combined with the iterative deconvolution-based source wavelet estimation procedure that is partially able to account for the frequency-dependent effects through an "effective" wavelet, performs remarkably well in weakly to moderately dispersive environments and has the ability to provide adequate tomographic reconstructions.

Expression and one-step purification of Plasmodium proteins in dictyostelium.

Nearly full-length Circumsporozoite protein (CSP) from Plasmodium falciparum, the C-terminal fragments from both P. falciparm and P. yoelii CSP and a fragment comprising 351 amino acids of P.vivax MSPI were expressed in the slime mold Dictyostelium discoideum. Discoidin-tag expression vectors allowed both high yields of these proteins and their purification by a nearly single-step procedure. We exploited the galactose binding activity of Discoidin Ia to separate the fusion proteins by affinity chromatography on Sepharose-4B columns. Inclusion of a thrombin recognition site allowed cleavage of the Discoidin-tag from the fusion protein. Partial secretion of the protein was obtained via an ER independent pathway, whereas routing the recombinant proteins to the ER resulted in glycosylation and retention. Yields of proteins ranged from 0.08 to 3 mg l(-1) depending on the protein sequence and the purification conditions. The recognition of purified MSPI by sera from P. vivax malaria patients was used to confirm the native conformation of the protein expressed in Dictyostelium. The simple purification procedure described here, based on Sepharose-4B, should facilitate the expression and the large-scale purification of various Plasmodium polypeptides.

Gliotransmission in the hippocampus, mechanisms and interaction with synaptic functions

SUMMARYAstrocytes represent the largest cell population in the human brain. In addition to a well established role as metabolic support for neuronal activity, in the last years these cells have been found to accomplish other important and, sometimes, unexpected functions. The tight enwrapping of synapses by astrocytic processes and the predominant expression of glutamate uptake carriers in the astrocytic rather than neuronal plasma membranes brought to the definition of a critical involvement of astrocytes in the clearance of glutamate from synaptic junctions. Moreover, several publications showed that astrocytes are able to release chemical transmitters (gliotransmitters) suggesting their active implication in the control of synaptic functions. Among gliotransmitters, the best characterized is glutamate, which has been proposed to be released from astrocytes in a Ca2+ dependent manner via exocytosis of synaptic-like microvesicles.In my thesis I present results leading to substantial advancement of the understanding of the mechanisms by which astrocytes modulate synaptic activity in the hippocampus, notably at excitatory synapses on dentate granule cells. I show that tumor necrosis factor- alpha (TNFa), a molecule that is generally involved in immune system functions, critically controls astrocyte-to-synapse communication (gliotransmission) in the brain. With constitutive levels of TNFa present, activation of purinergic G protein-coupled receptors in astrocytes, called P2Y1 receptors, induces localized intracellular calcium ([Ca2+]j) elevation in astrocytic processes (measured by two-photon microscopy) followed by glutamate release and activation of pre-synaptic NMDA receptors resulting in synaptic potentiation. In preparations lacking TNFa, astrocytes respond with identical [Ca2+]i elevations but fail to induce neuromodulation. I find that TNFa specifically controls the glutamate release step of gliotransmission. Addition of very low (picomolar) TNFa concentrations to preparations lacking the cytokine, promptly reconstitutes both normal exocytosis in cultured astrocytes and gliotransmission in hippocampal slices. These data provide the first demonstration that gliotransmission and its synaptic effects are controlled not only by astrocyte [Ca2+]i elevations but also by permissive/homeostatic factors like TNFa.In addition, I find that higher and presumably pathological TNFa concentrations do not act just permissively but instead become direct and potent triggers of glutamate release from astrocytes, leading to a strong enhancement of excitatory synaptic activity. The TNFa action, like the one observed upon P2Y1R activation, is mediated by pre-synaptic NMDA receptors, but in this case the effect is long-lasting, and not reversible. Moreover, I report that a necessary molecular target for this action of TNFa is TNFR1, one of the two specific receptors for the cytokine, as I found that TNFa was unable to induce synaptic potentiation when applied in slices from TNFR1 knock-out (Tnfrlv") mice. I then created a double transgenic mouse model where TNFR1 is knocked out in all cells but can be re-expressed selectively in astrocytes and I report that activation of the receptors in these cells is sufficient to reestablish TNFa-dependent long-lasting potentiation of synaptic activity in the TNFR1 knock-out mice.I therefore discovered that TNFa is a primary molecule displaying both permissive and instructive roles on gliotransmission controlling synaptic functions. These reports might have profound implications for the understanding of both physiological and pathological processes associated to TNFa production, including inflammatory processes in the brain.RÉSUMÉLes astrocytes sont les cellules les plus abondantes du cerveau humain. Outre leur rôle bien établi dans le support métabolique de l'activité neuronale, d'autres fonctions importantes, et parfois inattendues de ces cellules ont été mises en lumière au cours de ces dernières années. Les astrocytes entourent étroitement les synapses de leurs fins processus qui expriment fortement les transporteurs du glutamate et permettent ainsi aux astrocytes de jouer un rôle critique dans l'élimination du glutamate de la fente synaptique. Néanmoins, les astrocytes semblent être capables de jouer un rôle plus intégratif en modulant l'activité synaptique, notamment par la libération de transmetteurs (gliotransmetteurs). Le gliotransmetteur le plus étudié est le glutamate qui est libéré par l'exocytose régulée de petites vésicules ressemblant aux vésicules synaptiques (SLMVs) via un mécanisme dépendant du calcium.Les résultats présentés dans cette thèse permettent une avancée significative dans la compréhension du mode de communication de ces cellules et de leur implication dans la transmission de l'information synaptique dans l'hippocampe, notamment des synapses excitatrices des cellules granulaires du gyrus dentelé. J'ai pu montrer que le « facteur de nécrose tumorale alpha » (TNFa), une cytokine communément associée au système immunitaire, est aussi fondamentale pour la communication entre astrocyte et synapse. Lorsqu'un niveau constitutif très bas de TNFa est présent, l'activation des récepteurs purinergiques P2Y1 (des récepteurs couplés à protéine G) produit une augmentation locale de calcium (mesurée en microscopie bi-photonique) dans l'astrocyte. Cette dernière déclenche ensuite une libération de glutamate par les astrocytes conduisant à l'activation de récepteurs NMDA présynaptiques et à une augmentation de l'activité synaptique. En revanche, dans la souris TNFa knock-out cette modulation de l'activité synaptique par les astrocytes n'est pas bien qu'ils présentent toujours une excitabilité calcique normale. Nous avons démontré que le TNFa contrôle spécifiquement l'exocytose régulée des SLMVs astrocytaires en permettant la fusion synchrone de ces vésicules et la libération de glutamate à destination des récepteurs neuronaux. Ainsi, nous avons, pour la première fois, prouvé que la modulation de l'activité synaptique par l'astrocyte nécessite, pour fonctionner correctement, des facteurs « permissifs » comme le TNFa, agissant sur le mode de sécrétion du glutamate astrocytaire.J'ai pu, en outre, démontrer que le TNFa, à des concentrations plus élevées (celles que l'on peut observer lors de conditions pathologiques) provoque une très forte augmentation de l'activité synaptique, agissant non plus comme simple facteur permissif mais bien comme déclencheur de la gliotransmission. Le TNFa provoque 1'activation des récepteurs NMD A pré-synaptiques (comme dans le cas des P2Y1R) mais son effet est à long terme et irréversible. J'ai découvert que le TNFa active le récepteur TNFR1, un des deux récepteurs spécifiques pour le TNFa. Ainsi, l'application de cette cytokine sur une tranche de cerveau de souris TNFR1 knock-out ne produit aucune modification de l'activité synaptique. Pour vérifier l'implication des astrocytes dans ce processus, j'ai ensuite mis au point un modèle animal doublement transgénique qui exprime le TNFR1 uniquement dans les astrocytes. Ce dernier m'a permis de prouver que l'activation des récepteurs TNFR1 astrocytaires est suffisante pour induire une augmentation de l'activité synaptique de manière durable.Nous avons donc découvert que le TNFa possède un double rôle, à la fois un rôle permissif et actif, dans le contrôle de la gliotransmission et, par conséquent, dans la modulation de l'activité synaptique. Cette découverte peut potentiellement être d'une extrême importance pour la compréhension des mécanismes physiologiques et pathologiques associés à la production du TNFa, en particulier lors de conditions inflammatoires.RÉSUMÉ GRAND PUBLICLes astrocytes représentent la population la plus nombreuse de cellules dans le cerveau humain. On sait, néanmoins, très peu de choses sur leurs fonctions. Pendant très longtemps, les astrocytes ont uniquement été considérés comme la colle du cerveau, un substrat inerte permettant seulement de lier les cellules neuronales entre elles. Il n'y a que depuis peu que l'on a découvert de nouvelles implications de ces cellules dans le fonctionnement cérébral, comme, entre autres, une fonction de support métabolique de l'activité neuronale et un rôle dans la modulation de la neurotransmission. C'est ce dernier aspect qui fait l'objet de mon projet de thèse.Nous avons découvert que l'activité des synapses (régions qui permettent la communication d'un neurone à un autre) qui peut être potentialisée par la libération du glutamate par les astrocytes, ne peut l'être que dans des conditions astrocytaires très particulières. Nous avons, en particulier, identifié une molécule, le facteur de nécrose tumorale alpha (TNFa) qui joue un rôle critique dans cette libération de glutamate astrocytaire.Le TNFa est surtout connu pour son rôle dans le système immunitaire et le fait qu'il est massivement libéré lors de processus inflammatoires. Nous avons découvert qu'en concentration minime, correspondant à sa concentration basale, le TNFa peut néanmoins exercer un rôle indispensable en permettant la communication entre l'astrocyte et le neurone. Ce mode de fonctionnement est assez probablement représentatif d'un processus physiologique qui permet d'intégrer la communication astrocyte/neurone au fonctionnement général du cerveau. Par ailleurs, nous avons également démontré qu'en quantité plus importante, le TNFa change son mode de fonctionnement et agit comme un stimulateur direct de la libération de glutamate par l'astrocyte et induit une activation persistante de l'activité synaptique. Ce mode de fonctionnement est assez probablement représentatif d'un processus pathologique.Nous sommes également arrivés à ces conclusions grâce à la mise en place d'une nouvelle souche de souris doublement transgéniques dans lesquelles seuls les astrocytes (etnon les neurones ou les autres cellules cérébrales) sont capables d'être activés par le TNFa.

Development of improved soluble inhibitors of FasL and CD40L based on oligomerized receptors.

TNF receptor family members fused to the constant domain of immunoglobulin G have been widely used as immunoadhesins in basic in vitro and in vivo research and in some clinical applications. In this study, we assemble soluble, high avidity chimeric receptors on a pentameric scaffold derived from the coiled-coil domain of cartilage oligomeric matrix protein (COMP). The affinity of Fas and CD40 (but not TNFR-1 and TRAIL-R2) to their ligands is increased by fusion to COMP, when compared to the respective Fc chimeras. In functional assays, Fas:COMP was at least 20-fold more active than Fas:Fc at inhibiting the action of sFasL, and CD40:COMP could block CD40L-mediated proliferation of B cells, whereas CD40:Fc could not. In conclusion, members of the TNF receptor family can display high specificity and excellent avidity for their ligands if they are adequately multimerized.

The sigma factor AlgU (AlgT) controls exopolysaccharide production and tolerance towards desiccation and osmotic stress in the biocontrol agent Pseudomonas fluorescens CHA0.

A variety of stress situations may affect the activity and survival of plant-beneficial pseudomonads added to soil to control root diseases. This study focused on the roles of the sigma factor AlgU (synonyms, AlgT, RpoE, and sigma(22)) and the anti-sigma factor MucA in stress adaptation of the biocontrol agent Pseudomonas fluorescens CHA0. The algU-mucA-mucB gene cluster of strain CHA0 was similar to that of the pathogens Pseudomonas aeruginosa and Pseudomonas syringae. Strain CHA0 is naturally nonmucoid, whereas a mucA deletion mutant or algU-overexpressing strains were highly mucoid due to exopolysaccharide overproduction. Mucoidy strictly depended on the global regulator GacA. An algU deletion mutant was significantly more sensitive to osmotic stress than the wild-type CHA0 strain and the mucA mutant were. Expression of an algU'-'lacZ reporter fusion was induced severalfold in the wild type and in the mucA mutant upon exposure to osmotic stress, whereas a lower, noninducible level of expression was observed in the algU mutant. Overexpression of algU did not enhance tolerance towards osmotic stress. AlgU was found to be essential for tolerance of P. fluorescens towards desiccation stress in a sterile vermiculite-sand mixture and in a natural sandy loam soil. The size of the population of the algU mutant declined much more rapidly than the size of the wild-type population at soil water contents below 5%. In contrast to its role in pathogenic pseudomonads, AlgU did not contribute to tolerance of P. fluorescens towards oxidative and heat stress. In conclusion, AlgU is a crucial determinant in the adaptation of P. fluorescens to dry conditions and hyperosmolarity, two major stress factors that limit bacterial survival in the environment.

MP2RAGE, a self bias-field corrected sequence for improved segmentation and T1-mapping at high field.

The large spatial inhomogeneity in transmit B(1) field (B(1)(+)) observable in human MR images at high static magnetic fields (B(0)) severely impairs image quality. To overcome this effect in brain T(1)-weighted images, the MPRAGE sequence was modified to generate two different images at different inversion times, MP2RAGE. By combining the two images in a novel fashion, it was possible to create T(1)-weighted images where the result image was free of proton density contrast, T(2) contrast, reception bias field, and, to first order, transmit field inhomogeneity. MP2RAGE sequence parameters were optimized using Bloch equations to maximize contrast-to-noise ratio per unit of time between brain tissues and minimize the effect of B(1)(+) variations through space. Images of high anatomical quality and excellent brain tissue differentiation suitable for applications such as segmentation and voxel-based morphometry were obtained at 3 and 7 T. From such T(1)-weighted images, acquired within 12 min, high-resolution 3D T(1) maps were routinely calculated at 7 T with sub-millimeter voxel resolution (0.65-0.85 mm isotropic). T(1) maps were validated in phantom experiments. In humans, the T(1) values obtained at 7 T were 1.15+/-0.06 s for white matter (WM) and 1.92+/-0.16 s for grey matter (GM), in good agreement with literature values obtained at lower spatial resolution. At 3 T, where whole-brain acquisitions with 1 mm isotropic voxels were acquired in 8 min, the T(1) values obtained (0.81+/-0.03 s for WM and 1.35+/-0.05 for GM) were once again found to be in very good agreement with values in the literature.

Use of an empty, Plasmapore-covered titanium cage for interbody fusion after anterior cervical microdiscectomy.

The use of cages of different material and shapes for cervical discectomy with fusion (ACDF) has increased during the last few years. The use of additional osteogenic material is controversial. We prospectively evaluated an empty, Plasmapore-covered titanium cage (PCTC) in 45 patients undergoing 58 ACDFs. Patients were evaluated using standard clinical and radiological criteria. Good to excellent outcome was achieved in 93%, 78% and 75% after 3, 12 and 48 months, respectively. Sixty-five percent of patients could resume their prior work after 48 months. Disc space height and lordosis could be preserved in all cases. Two percent of the treated levels showed subsidence and 2% increased segmental motion. There were no procedure-related complications. Implantation of an empty PCTC after microsurgical anterior cervical discectomy is a safe procedure with good results and low incidence of complications. Disc height and lordosis can be preserved with low incidence of subsidence and good fusion rates.

Purification and ligand binding of a soluble class I major histocompatibility complex molecule consisting of the first three domains of H-2Kd fused to beta 2-microglobulin expressed in the baculovirus-insect cell system.

A recombinant baculovirus encoding a single-chain murine major histocompatibility complex class I molecule in which the first three domains of H-2Kd are fused to beta 2-microglobulin (beta 2-m) via a 15-amino acid linker has been isolated and used to infect lepidopteran cells. A soluble, 391-amino acid single-chain H-2Kd (SC-Kd) molecule of 48 kDa was synthesized and glycosylated in insect cells and could be purified in the absence of detergents by affinity chromatography using the anti-H-2Kd monoclonal antibody SF1.1.1.1. We tested the ability of SC-Kd to bind antigenic peptides using a direct binding assay based on photoaffinity labeling. The photoreactive derivative was prepared from the H-2Kd-restricted Plasmodium berghei circumsporozoite protein (P.b. CS) peptide 253-260 (YIPSAEKI), a probe that we had previously shown to be unable to bind to the H-2Kd heavy chain in infected cells in the absence of co-expressed beta 2-microglobulin. SC-Kd expressed in insect cells did not require additional mouse beta 2-m to bind the photoprobe, indicating that the covalently attached beta 2-m could substitute for the free molecule. Similarly, binding of the P.b. CS photoaffinity probe to the purified SC-Kd molecule was unaffected by the addition of exogenous beta 2-m. This is in contrast to H-2KdQ10, a soluble H-2Kd molecule in which beta 2-m is noncovalently bound to the soluble heavy chain, whose ability to bind the photoaffinity probe is greatly enhanced in the presence of an excess of exogenous beta 2-m. The binding of the probe to SC-Kd was allele-specific, since labeling was selectively inhibited only by antigenic peptides known to be presented by the H-2Kd molecule.

Functional interactions between the estrogen receptor and the transcription activator Sp1 regulate the estrogen-dependent transcriptional activity of the vitellogenin A1 io promoter.

Two distinct, TATA box-containing promoters regulate the transcriptional activity of the Xenopus vitellogenin A1 gene. These two promoters are of different strength and are separated by 1.8 kilobase pairs of untranslated sequence. Estrogen receptor (ER) and its ligand, 17beta-estradiol, induce the activity of both promoters. The estrogen response elements (EREs) are located proximal to the downstream i promoter while no ERE-like sequences have been identified in the vicinity of the upstream io promoter. We show here, that transcriptional activity of the upstream io promoter is Sp1-dependent. Moreover, we demonstrate that estrogen inducibility of the io promoter results from functional interactions between the io bound Sp1 and the ER bound at the proximity of i. Functional interactions between Sp1 and ER do not require the presence of a TATA box for transcriptional activation, as is demonstrated using the acyl-CoA oxidase promoter. The relative positions that ER and Sp1 occupy with respect to the initiation site determines whether these two transcription activators can synergize for transcription initiation.

Noninvasive coronary vessel wall and plaque imaging with magnetic resonance imaging.

BACKGROUND: Conventional x-ray angiography frequently underestimates the true burden of atherosclerosis. Although intravascular ultrasound allows for imaging of coronary plaque, this invasive technique is inappropriate for screening or serial examinations. We therefore sought to develop a noninvasive free-breathing MR technique for coronary vessel wall imaging. We hypothesized that such an approach would allow for in vivo imaging of coronary atherosclerosis. METHODS AND RESULTS: Ten subjects, including 5 healthy adult volunteers (aged 35+/-17 years, range 19 to 56 years) and 5 patients (aged 60+/-4 years, range 56 to 66 years) with x-ray-confirmed coronary artery disease (CAD), were studied with a T2-weighted, dual-inversion, fast spin-echo MR sequence. Multiple adjacent 5-mm cross-sectional images of the proximal right coronary artery were obtained with an in-plane resolution of 0.5x1.0 mm. A right hemidiaphragmatic navigator was used to facilitate free-breathing MR acquisition. Coronary vessel wall images were readily acquired in all subjects. Both coronary vessel wall thickness (1.5+/-0.2 versus 1.0+/-0.2 mm) and wall area (21.2+/-3.1 versus 13.7+/-4.2 mm(2)) were greater in patients with CAD (both P:<0.02 versus healthy adults). CONCLUSIONS: In vivo free-breathing coronary vessel wall and plaque imaging with MR has been successfully implemented in humans. Coronary wall thickness and wall area were significantly greater in patients with angiographic CAD. The presented technique may have potential applications in patients with known or suspected atherosclerotic CAD or for serial evaluation after pharmacological intervention.

EWS-FLI-1 modulates miRNA145 and SOX2 expression to initiate mesenchymal stem cell reprogramming toward Ewing sarcoma cancer stem cells.

Cancer stem cells (CSCs) display plasticity and self-renewal properties reminiscent of normal tissue stem cells, but the events responsible for their emergence remain obscure. We recently identified CSCs in Ewing sarcoma family tumors (ESFTs) and showed that they retain mesenchymal stem cell (MSC) plasticity. In the present study, we addressed the mechanisms that underlie ESFT CSC development. We show that the EWS-FLI-1 fusion gene, associated with 85%-90% of ESFTs and believed to initiate their pathogenesis, induces expression of the embryonic stem cell (ESC) genes OCT4, SOX2, and NANOG in human pediatric MSCs (hpMSCs) but not in their adult counterparts. Moreover, under appropriate culture conditions, hpMSCs expressing EWS-FLI-1 generate a cell subpopulation displaying ESFT CSC features in vitro. We further demonstrate that induction of the ESFT CSC phenotype is the result of the combined effect of EWS-FLI-1 on its target gene expression and repression of microRNA-145 (miRNA145) promoter activity. Finally, we provide evidence that EWS-FLI-1 and miRNA-145 function in a mutually repressive feedback loop and identify their common target gene, SOX2, in addition to miRNA145 itself, as key players in ESFT cell differentiation and tumorigenicity. Our observations provide insight for the first time into the mechanisms whereby a single oncogene can reprogram primary cells to display a CSC phenotype.