The detection of Helicobacter pylori in paraffin sections using the PCR technique and various primers as compared to histological techniques

Helicobacter pylori is thought to represent a significant etiopathogenic factor in diseases of the upper gastrointestinal tract. It seems, therefore, important to elaborate effective techniques for its detection. The aim of the present study was to evaluate the effectiveness of Helicobacter pylori detection using the PCR technique on paraffin sections with various pairs of primers and to compare the results with those of a histological appraisal. Material for the studies involved 50 paraffin blocks with gastric mucosa biopsies fixed in 4% buffered formalin. In this material 4 tests were performed with the aim of diagnosing Helicobacter pylori infection: 1) H+E staining, 2) staining by the Giemsa technique, 3) an immunocytochemical technique with antibodies against H. pylori and 4) the PCR technique with various primers. In the present study the most reliable results for H. pylori detection as well as the most pronounced correlation were obtained by using the PCR technique with primers for the ureC gene, immunohistochemistry and staining according to Giemsa. Less compatible results were obtained employing the two PCR techniques which utilise various primers. The experiments confirmed the usefulness of the PCR technique in the detection of Helicobacter pylori in paraffin sections by using a suitable pair of primers, and also indicated that Giemsa staining and immunohistochemistry should be taken into account

Visualization of class A GPCR oligomerization by image-based fluorescence fluctuation spectroscopy

G protein-coupled receptors (GPCRs) represent the largest class of cell surface receptors conveying extracellular information into intracellular signals. Many GPCRs have been shown to be able to oligomerize and it is firmly established that Class C GPCRs (e.g. metabotropic glutamate receptors) function as obligate dimers. However, the oligomerization capability of the larger Class A GPCRs (e.g. comprising the β-adrenergic receptors (β-ARs)) is still, despite decades of research, highly debated. Here we assess the oligomerization behavior of three prototypical Class A GPCRs, the β1-ARs, β2-ARs, and muscarinic M2Rs in single, intact cells. We combine two image correlation spectroscopy methods based on molecular brightness, i.e. the analysis of fluorescence fluctuations over space and over time, and thereby provide an assay able to robustly and precisely quantify the degree of oligomerization of GPCRs. In addition, we provide a comparison between two labelling strategies, namely C-terminally-attached fluorescent proteins and N-terminally-attached SNAP-tags, in order to rule out effects arising from potential fluorescent protein-driven oligomerization. The degree of GPCR oligomerization is expressed with respect to a set of previously reported as well as newly established monomeric or dimeric control constructs. Our data reveal that all three prototypical GPRCs studied display, under unstimulated conditions, a prevalently monomeric fingerprint. Only the β2-AR shows a slight degree of oligomerization. From a methodological point of view, our study suggests three key aspects. First, the combination of two image correlation spectroscopy methods allows addressing cells transiently expressing high concentrations of membrane receptors, far from the single molecule regime, at a density where the kinetic equilibrium should favor dimers and higher-order oligomers. Second, our methodological approach, allows to selectively target cell membrane regions devoid of artificial oligomerization hot-spots (such as vesicles). Third, our data suggest that the β1-AR appears to be a superior monomeric control than the widely used membrane protein CD86. Taken together, we suggest that our combined image correlation spectroscopy method is a powerful approach to assess the oligomerization behavior of GPCRs in intact cells at high expression levels

Pentoxifylline inhibits the fibrogenic activity of pleural effusions and transforming growth factor-β

Physiopathology of organ fibrosis is far from being completely understood, and the efficacy of the available therapeutic strategies is disappointing. We chose pleural disease for further studies and addressed the questions of which cytokines are relevant in pleural fibrosis and which drugs might interrupt its development. We screened pleural effusions for mediators thought to interfere with fibrogenesis (transforming growth factor-β (TGF-β), tumour necrosis factor α (TNFα), soluble TNF-receptor p55 (sTNF-R)) and correlated the results with patient clinical outcome in terms of extent of pleural thickenings. We found pleural thickenings correlated with TGF-β (p < 0.005) whereas no correlations could be observed with TNFα and sTNF-R. Further, we were interested in finding out how TGF-β effects on fibroblast growth could be modulated. We found that pentoxifylline is able to inhibit both fibroblast proliferation and collagen synthesis independently of the stimulus. We conclude that, judging from in vitro studies, pentoxifylline might offer a new approach in the therapy of pleural as well as pulmonary fibrosis

A universal bioluminescence resonance energy transfer sensor design enables high-sensitivity screening of GPCR activation dynamics

G-protein-coupled receptors (GPCRs) represent one of the most important classes of drug targets. The discovery of new GCPR therapeutics would greatly benefit from the development of a generalizable high-throughput assay to directly monitor their activation or de-activation. Here we screened a variety of labels inserted into the third intracellular loop and the C-terminus of the α-adrenergic receptor and used fluorescence (FRET) and bioluminescence resonance energy transfer (BRET) to monitor ligand-binding and activation dynamics. We then developed a universal intramolecular BRET receptor sensor design to quantify efficacy and potency of GPCR ligands in intact cells and real time. We demonstrate the transferability of the sensor design by cloning β-adrenergic and PTH1-receptor BRET sensors and monitored their efficacy and potency. For all biosensors, the Z factors were well above 0.5 showing the suitability of such design for microtiter plate assays. This technology will aid the identification of novel types of GPCR ligands

Preparation and structural properties of thin films and multilayers of the Heusler compounds Cu2MnAl, Co2MnSn, Co2MnSi and Co2MnGe

We report on the preparation of thin films and multilayers of the intermetallic Heusler compound CuMnAl, Co2MnSn, Co2MnSi and Co2MnGe by rf-sputtering on MgO and Al2O3 substrates. Cu2MnAl can be grown epitaxially with (100)-orientation on MgO (100) and in (110)-orientation on Al2O3 a-plane. The Co based Heusler alloys need metallic seedlayers to induce high quality textured growth. We also have prepared multilayers with smooth interfaces by combining the Heusler compounds with Au and V. An analysis of the ferromagnetic saturation magnetization of the films indicates that the Cu2MnAl-compound tends to grow in the disordered B2-type structure whereas the Co-based Heusler alloy thin films grow in the ordered L21 structure. All multilayers with thin layers of the Heusler compounds exhibit a definitely reduced ferromagnetic magnetization indicating substantial disorder and intermixing at the interfaces.Comment: 21 pages, 8 figure

Development of a conformational histamine H(3) receptor biosensor for the synchronous screening of agonists and inverse agonists

The histamine H(3) receptor (H(3)R) represents a highly attractive drug target for the treatment of various central nervous system disorders, but the discovery of novel H(3)R targeting compounds relies on the assessment of highly amplified intracellular signaling events that do not only reflect H(3)R modulation and carry the risk of high false-positive and -negative screening rates. To address these limitations, we designed an intramolecular H(3)R biosensor based on the principle of bioluminescence resonance energy transfer (BRET) that reports the receptor's real-time conformational dynamics and provides an advanced tool to screen for both H(3)R agonists and inverse agonists in a live cell screening-compatible assay format. This conformational G-protein-coupled receptor (GPCR) sensor allowed us to characterize the pharmacological properties of known and new H(3) receptor ligands with unprecedented accuracy. Interestingly, we found that one newly developed H(3) receptor ligand possesses even stronger inverse agonistic activity than reference H(3)R inverse agonists including the current gold standard pitolisant. Taken together, we describe here the design and validation of the first screening-compatible H(3)R conformational biosensor that will aid in the discovery of novel H(3)R ligands and can be employed to gain deeper insights into the (in-)activation mechanism of this highly attractive drug target

Stepwise activation of a class C GPCR begins with millisecond dimer rearrangement

G protein-coupled receptors (GPCRs) are key biological switches that transmit both internal and external stimuli into the cell interior. Among the GPCRs, the "light receptor" rhodopsin has been shown to activate with a rearrangement of the transmembrane (TM) helix bundle within ~1 ms, while all other receptors are thought to become activated within ~50 ms to seconds at saturating concentrations. Here, we investigate synchronous stimulation of a dimeric GPCR, the metabotropic glutamate receptor type 1 (mGluR1), by two entirely different methods: (i) UV light-triggered uncaging of glutamate in intact cells or (ii) piezo-driven solution exchange in outside-out patches. Submillisecond FRET recordings between labels at intracellular receptor sites were used to record conformational changes in the mGluR1. At millimolar ligand concentrations, the initial rearrangement between the mGluR1 subunits occurs at a speed of τ(1) ~ 1-2 ms and requires the occupancy of both binding sites in the mGluR1 dimer. These rapid changes were followed by significantly slower conformational changes in the TM domain (τ(2) ~ 20 ms). Receptor deactivation occurred with time constants of ~40 and ~900 ms for the inter- and intrasubunit conformational changes, respectively. Together, these data show that, at high glutamate concentrations, the initial intersubunit activation of mGluR1 proceeds with millisecond speed, that there is loose coupling between this initial step and activation of the TM domain, and that activation and deactivation follow a cyclic pathway, including-in addition to the inactive and active states-at least two metastable intermediate states

Hyper-domains in exchange bias micro-stripe pattern

A combination of experimental techniques, e.g. vector-MOKE magnetometry, Kerr microscopy and polarized neutron reflectometry, was applied to study the field induced evolution of the magnetization distribution over a periodic pattern of alternating exchange bias (EB) stripes. The lateral structure is imprinted into a continuous ferromagnetic/antiferromagnetic EB bilayer via laterally selective exposure to He-ion irradiation in an applied field. This creates an alternating frozen-in interfacial EB field competing with the external field in the course of the re-magnetization. It was found that in a magnetic field applied at an angle with respect to the EB axis parallel to the stripes the re-magnetization process proceeds via a variety of different stages. They include coherent rotation of magnetization towards the EB axis, precipitation of small random (ripple) domains, formation of a stripe-like alternation of the magnetization, and development of a state in which the magnetization forms large hyper-domains comprising a number of stripes. Each of those magnetic states is quantitatively characterized via the comprehensive analysis of data on specular and off-specular polarized neutron reflectivity. The results are discussed within a phenomenological model containing a few parameters, which can readily be controlled by designing systems with a desired configuration of magnetic moments of micro- and nano-elements