A compact von Hámos spectrometer for parallel X-ray Raman scattering and X-ray emission spectroscopy at ID20 of the European Synchrotron Radiation Facility.

A compact spectrometer for medium-resolution resonant and non-resonant X-ray emission spectroscopy in von Hámos geometry is described. The main motivation for the design and construction of the spectrometer is to allow for acquisition of non-resonant X-ray emission spectra while measuring non-resonant X-ray Raman scattering spectra at beamline ID20 of the European Synchrotron Radiation Facility. Technical details are provided and the performance and possible use of the spectrometer are demonstrated by presenting results of several X-ray spectroscopic methods on various compounds.

A high-energy-resolution resonant inelastic X-ray scattering spectrometer at ID20 of the European Synchrotron Radiation Facility.

An end-station for resonant inelastic X-ray scattering and (resonant) X-ray emission spectroscopy at beamline ID20 of ESRF - The European Synchrotron is presented. The spectrometer hosts five crystal analysers in Rowland geometry for large solid angle collection and is mounted on a rotatable arm for scattering in both the horizontal and vertical planes. The spectrometer is optimized for high-energy-resolution applications, including partial fluorescence yield or high-energy-resolution fluorescence detected X-ray absorption spectroscopy and the study of elementary electronic excitations in solids. In addition, it can be used for non-resonant inelastic X-ray scattering measurements of valence electron excitations.

A miniature closed-circle flow cell for high photon flux X-ray scattering experiments.

A closed-circle miniature flow cell for high X-ray photon flux experiments on radiation-sensitive liquid samples is presented. The compact cell is made from highly inert material and the flow is induced by a rotating magnetic stir bar, which acts as a centrifugal pump inside the cell. The cell is ideal for radiation-sensitive yet precious or hazardous liquid samples, such as concentrated acids or bases. As a demonstration of the cell's capabilities, X-ray Raman scattering spectroscopy data on the oxygen K-edge of liquid water under ambient conditions are presented.

Metabolic intervention on lipid synthesis converging pathways abrogates prostate cancer growth.

One of the most conserved features of all cancers is a profound reprogramming of cellular metabolism, favoring biosynthetic processes and limiting catalytic processes. With the acquired knowledge of some of these important changes, we have designed a combination therapy in order to force cancer cells to use a particular metabolic pathway that ultimately results in the accumulation of toxic products. This innovative approach consists of blocking lipid synthesis, at the same time that we force the cell, through the inhibition of AMP-activated kinase, to accumulate toxic intermediates, such as malonyl-coenzyme A (malonyl-CoA) or nicotinamide adenine dinucleotide phosphate. This results in excess of oxidative stress and cancer cell death. Our new therapeutic strategy, based on the manipulation of metabolic pathways, will certainly set up the basis for new upcoming studies defining a new paradigm of cancer treatment.

Histamine H1-receptor antagonists inhibit nuclear factor-kappaB and activator protein-1 activities via H1-receptor-dependent and -independent mechanisms.

BACKGROUND: Histamine H1-receptor antagonists are used to relieve the symptoms of an immediate allergic reaction. They have additional anti-inflammatory effects that could result from an inhibition of the transcription factors activator protein-1 (AP-1) and nuclear factor-kappa B (NF-kappaB). The implication of the H1-receptor in these effects is controversial. Diphenhydramine is a first-generation H1-receptor antagonist while mizolastine and desloratadine are second-generation compounds. Mizolastine is also an inhibitor of 5-lipoxygenase (5-LO), an enzyme that has been involved in NF-kappaB activation. OBJECTIVE: We measured the ability of antihistamines to reverse histamine-induced smooth muscle contraction, an effect that involves the H1-receptor. We then investigated whether these drugs affect NF-kappaB and AP-1 activities in A549 lung epithelial cells, and whether this potential regulation involves H1-receptor and 5-LO. METHODS: Muscle tone was measured on tracheal segments of guinea-pigs. The H1-receptor was overexpressed by transfection and detected by Western blotting and immunofluorescence microscopy. NF-kappaB and AP-1 activities were assessed by reporter gene assays in cells overexpressing or not overexpressing the H1-receptor. Production of regulated upon activation, normal T cell expressed andsecreted (RANTES), a chemokine whose expression is induced through NF-kappaB, was measured using an immunoassay. RESULTS: H1-receptor antagonists reversed histamine-induced contraction in a dose-dependent manner. Induction of AP-1 and NF-kappaB activities by histamine and the down-regulatory effect of antihistamines required overexpression of the H1-receptor. In contrast, when tumour necrosis factor-alpha and a phorbol ester were used to stimulate NF-kappaB and AP-1 activities, respectively, repression of these activities did not involve the H1-receptor. Indeed, repression was triggered only by a subset of H1-receptor antagonists and was not stronger after overexpression of the H1-receptor. Mizolastine and desloratadine dose-dependently decreased tumour necrosis factor-alpha-induced production of RANTES. Diphenhydramine, H2- and H3-receptor antagonists as well as selective inhibitors of 5-LO were ineffective in this assay. CONCLUSION: Repression of NF-kappaB and AP-1 activities by H1-receptor antagonists involves H1-receptor-dependent and -independent mechanisms but not 5-LO.

Improving the performance of high-resolution X-ray spectrometers with position-sensitive pixel detectors.

A dispersion-compensation method to remove the cube-size effect from the resolution function of diced analyzer crystals using a position-sensitive two-dimensional pixel detector is presented. For demonstration, a resolution of 23 meV was achieved with a spectrometer based on a 1 m Rowland circle and a diced Si(555) analyzer crystal in a near-backscattering geometry, with a Bragg angle of 88.5 degrees . In this geometry the spectrometer equipped with a traditional position-insensitive detector provides a resolution of 190 meV. The dispersion-compensation method thus allows a substantial increase in the resolving power without any loss of signal intensity.

Fluticasone propionate and mometasone furoate have equivalent transcriptional potencies.

BACKGROUND: Glucocorticoids exert their anti-inflammatory effects mainly through transrepression of the transcription factors activator protein-1 (AP-1) and nuclear factor-kappa B (NF-kappaB). Certain adverse effects of glucocorticoids are mediated through gene transactivation. Fluticasone propionate (FP) and mometasone furoate (MF) are the most recently developed topical glucocorticoids for the treatment of airway disorders. Their relative capacities to repress AP-1 and NF-kappaB activities are not known and comparison of their transactivation potencies has given unclear results. OBJECTIVE: To determine the relative transactivation and transrepression potencies of FP and MF. METHODS: Transactivation assays were performed in HeLa cells carrying a glucocorticoid-inducible luciferase gene. To measure transrepressive potencies of FP and MF, A549 lung epithelial cells were transiently transfected with an AP-1- or NF-kappaB-dependent luciferase gene. Using an immunoassay, we also evaluated the ability of MF and FP to inhibit the production of Regulated upon Activation, Normal T-cell Expressed and Secreted (RANTES), a pro-inflammatory cytokine, whose gene is controlled by AP-1 and NF-kappaB. Areas under the dose-response curve were calculated to determine relative potencies. RESULTS: FP and MF are equipotent for transactivation. Both molecules show globally the same potency to inhibit AP-1 and NF-kappaB activities and RANTES production. MF and FP have very significant transcriptional effects at 2x10(-10) M, which is the peak concentration reached in the plasma after inhalation of high dosages. Indeed, they produce a 17-fold induction of luciferase in the transactivation assay, and inhibit AP-1 activity, NF-kappaB activity and RANTES release by approximately 40%. CONCLUSION: FP and MF have the same ability to trigger gene activation and also the same potency to inhibit AP-1 and NF-kappaB activities. Their strong transcriptional effects at 2x10(-10) M suggest that these compounds act not only topically but also systemically, with the risk of provoking concomitant adverse effects at high dosages.

Correlation between different gene expression assays designed to measure trans-activation potencies of systemic glucocorticoids.

The glucocorticoids (GC) betamethasone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone and triamcinolone acetonide are currently used in the treatment of inflammatory diseases. Through a process called trans-activation, GC activate gene expression and produce various physiological and pharmacological effects. In particular, by inducing gluconeogenic enzymes, long-term GC treatment may cause diabetes. Using three different assays, we have extensively compared the capacity of the above GC to activate gene expression. trans-Activation of a GC inducible luciferase gene was assessed in HeLa and A549 cells after stable and transient transfection, respectively. In hepatoma tissue culture cells, we measured trans-activation of the endogenous gene encoding tyrosine aminotransferase, a gluconeogenic enzyme. Half-maximal effective concentrations of GC were determined by dose-response analyses. Results obtained with these assays were highly correlated and GC were ranked in three groups according to their trans-activation potency: betamethasone, dexamethasone, and triamcinolone acetonide > methylprednisolone and prednisolone > hydrocortisone. Potencies were not strictly related to receptor binding affinities and not significantly affected by the amount of endogenous GC receptor.

Tissue inhibitor of metalloproteinase-1 levels in bronchoalveolar lavage fluid from asthmatic subjects.

Airway remodeling is a well-recognized feature in patients with chronic asthma. The accumulation in the submucosa of fibrous proteins that are substrates of matrix metalloproteinases (MMP), and the demonstration of increased levels of MMP-9 in bronchoalveolar lavage fluid, prompted us to determine whether there was an imbalance between MMPs and tissue inhibitors of metalloproteinase (TIMP) in such patients. We investigated the presence of TIMPs and other MMPs. TIMP levels were compared with those of all MMPs and inflammatory cytokines. Adults with stable asthma, either untreated or treated with glucocorticoids (GCs), were enrolled. Healthy nonsmokers served as a control population. MMPs and TIMPs were identified through zymography or immunoblotting. TIMPs, MMPs, and cytokines were measured with enzyme immunoassays. TIMP-1 levels were significantly higher in untreated asthmatic subjects than in GC-treated subjects or controls (p < 0.0001), and were far greater than those of MMP-1, MMP-2, MMP-3, and MMP-9 combined. TIMP-2 was undetectable. TIMP-1 levels were correlated with levels of interleukin-6 (p < 0.012) and the number of alveolar macrophages recovered (p < 0.005). This observation has important implications, since an excess of TIMP-1 could lead to airway fibrosis, a hallmark of airway remodelling in patients with chronic asthma.

Increased expression of tissue inhibitor of metalloproteinase-1 and loss of correlation with matrix metalloproteinase-9 by macrophages in asthma.

Alveolar macrophages (AMs) can mediate tissue destruction and repair by synthesizing matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) as well as inflammatory cytokines, which regulate their production. Imbalances between these enzymes and inhibitors may contribute to the tissue damage and remodeling seen in inflammatory diseases. In this study, we examined the role of AMs in chronic asthma. We have previously demonstrated an increased production of MMP-9 by AMs in untreated asthmatic patients as compared with healthy subjects, and in asthmatics treated with inhaled corticosteroids and patients with chronic bronchitis. We now report on the expression of TIMP-1, the inhibitor of MMP-9, and compare the levels and the regulation by cytokines of both MMP-9 and TIMP-1. Enzyme and inhibitor were measured using an enzyme immunoassay and immunoprecipitation. TIMP-1 steady-state mRNA levels were measured using the RNase protection assay. AMs from untreated asthmatics were found to produce more TIMP-1 both at protein and mRNA levels than AMs from other groups. The release of TIMP-1 and MMP-9 from individual AMs was significantly correlated in control populations and the molecules mainly complexed to each other, whereas this was not true for untreated asthmatics, indicating an imbalance between MMP-9 and TIMP-1 production. In the latter population, TIMP-1 release was inhibited by an anti-IL-6 antibody and MMP-9 release by anti-TNF-alpha, anti-IL-6, and anti-IL-1/beta antibodies. The imbalance of MMP-9 and TIMP-1 production, via the involvement of different cytokines, suggests that AMs may be involved in the abnormal repair observed in chronic asthma.

Differential interaction of nuclear receptors with the putative human transcriptional coactivator hTIF1.

Hormonal regulation of gene activity is mediated by nuclear receptors acting as ligand-activated transcription factors. Intermediary factors interacting with their activation functions are required to mediate transcriptional stimulation. In search of such receptor interacting proteins, we have screened a human cDNA expression library and isolated a human protein that interacts in vitro with transcriptionally active estrogen receptors (ER). Sequence analysis reveals that this protein is the human homolog of mouse TIF1 (transcription intermediary factor 1) shown to enhance nuclear receptor ligand-dependent activation function 2 (AF2) in yeast. We have characterized the nuclear receptor binding site on hTIF1 and shown that a region of 26 residues is sufficient for hormone-dependent binding to the estrogen receptor. As shown by point mutagenesis, the AF2 activation domain of ER is required for the binding of hTIF1 but not sufficient, since a short region encompassing the conserved amphipathic alpha-helix corresponding to this domain fails to precipitate hTIF1. We also demonstrate that hTIF1 association with DNA-bound ER requires the presence of estradiol. Finally, we show that the interaction of hTIF1 with receptors is selective since strong in vitro hormone-dependent binding is only observed with some members of the nuclear receptor superfamily.