The novel inotropic agent CGP 48506 alters force primarily by Ca(2+)-independent mechanisms in porcine skinned trabeculae

OBJECTIVES: The aim was to determine whether, and by what mechanism(s), a novel inotropic agent 5-methyl-6-phenyl-1,3,5,6-tetrahydro-3, 6-methano-1,5-benzodiazocine-2,4-dione (BA 41899) and its enantiomers directly alter the Ca2+ sensitivity of cardiac myofilaments. METHODS: Porcine ventricular trabeculae were permeabilised with Triton X-100. The relationship between force and pCa (-log[Ca2+]) was determined in the presence and absence of ATP. Troponin I was extracted, using vanadate, to produce unregulated maximally activated myofilaments. Force and actomyosin ATPase activity were measured simultaneously to determine tension cost (ATPase activity/tension). The effects of the (+) enantiomer (CGP 48506) on the twitch of intact muscle were demonstrated using rat papillary muscle. RESULTS: 100 microM BA 41899 had a pronounced Ca2+ sensitising effect on force production by porcine skinned cardiac fibres, increasing the pCa required for 50% maximal activation by 0.64 units, while suppressing maximum force by 18.3%. Resting tension was unaffected. These actions were primarily caused by CGP 48506 and were concentration dependent. At concentrations less than 100 microM, CGP 48506 also increased twitch amplitude in intact papillary muscles with no effect on resting tension, whereas 100 microM CGP 48506 increased resting force due to a slowing of relaxation. 100 microM CGP 48506 potentiated Ca(2+)-independent rigor tension in skinned trabeculae, indicating a Ca2+ sensitising mechanism unrelated to Ca2+ binding to troponin C. Tension cost was unaffected by 100 microM CGP 48506 over the entire range of activating Ca2+ concentrations. Suppression of maximum force by CGP 48506 was independent of both Ca2+ concentration and the regulatory troponin complex. CONCLUSIONS: Both the increase in Ca2+ sensitivity during submaximal activation and the depression of maximum force which are induced by CGP 48506 in skinned trabeculae occur at least partly through Ca(2+)-independent mechanisms.     

3D Micro-PIXE at atmospheric pressure: A new tool for the investigation of art and archaeological objects

The paper describes a novel experiment characterized by the development of a confocal geometry in an external Micro-PIXE set-up. The position of X-ray optics in front of the X-ray detector and its proper alignment with respect to the proton micro-beam focus provided the possibility of carrying out 3D Micro-PIXE analysis. As a first application, depth intensity profiles of the major elements that compose the patina layer of a quaternary bronze alloy were measured. A simulation approach of the 3D Micro-PIXE data deduced elemental concentration profiles in rather good agreement with corresponding results obtained by electron probe micro-analysis from a cross-sectioned patina sample. With its non-destructive and depth-resolving properties, as well as its feasibility in atmospheric pressure, 3D Micro-PIXE seems especially suited for investigations in the field of cultural heritage.     

Negative chronotropic and inotropic effects exerted by diadenosine hexaphosphate (AP6A) via A1-adenosine receptors

1. Diadenosine hexaphosphate (AP6A) exerts vasoconstrictive effects. The purpose of this study was to investigate whether AP6A has any effect on cardiac function. 2. The effects of AP6A (0.1-100 microM) on cardiac contractility and frequency were studied in guinea-pig and human isolated cardiac preparations. Furthermore, the effects of AP6A on the amplitude of the L-type calcium current, on the adenosine 3':5'-cyclic monophosphate (cyclic AMP) content and on the phosphorylation of regulatory phosphoproteins, i.e. phospholamban and troponin inhibitor, were investigated in guinea-pig isolated ventricular myocytes. 3. In isolated spontaneously beating right atria of the guinea-pig AP6A exerted a negative chronotropic effect and reduced the rate of contraction maximally by 35% (IC20 = 35 microM). 4. In isolated electrically driven left atria of the guinea-pig AP6A exerted a negative inotropic effect and reduced force of contraction maximally by 23% (IC20 = 70 microM). 5. In isolated electrically driven papillary muscles of the guinea-pig AP6A alone was ineffective, but attenuated isoprenaline-stimulated force of contraction maximally by 23% (IC20 = 60 microM). Furthermore, AP6A attenuated the relaxant effect of isoprenaline. 6. In human isolated electrically driven ventricular preparations AP6A alone was ineffective, but attenuated isoprenaline-stimulated force of contraction by maximally 42% (IC20 = 18 microM). Moreover, AP6A attenuated the relaxant effect of isoprenaline. 7. All these effects of AP6A were abolished by the selective A1-adenosine receptor antagonist 1,3-dipropyl-cyclopentyl-xanthine (DPCPX, 0.3 microM), whereas the M-cholinoceptor antagonist atropine (10 microM) and the P2-purinoceptor antagonist suramin (300 microM) failed to abolish the effects of AP6A. 8. AP6A 100 microM had no effect on the amplitude of the L-type calcium current, but attenuated isoprenaline-stimulated L-type calcium current. The maximum of the current-voltage relationship (I-V curve) was shifted to the left by isoprenaline and additional application of AP6A shifted the I-V curve back to the right to the control value. The phosphorylation state of phospholamban and the troponin inhibitor was unchanged by AP6A alone, but was markedly attenuated by AP6A in the presence of isoprenaline. Cyclic AMP levels remained unchanged by AP6A, even after stimulation with isoprenaline. 9. In summary, AP6A exerts negative chronotropic and inotropic effects in guinea-pig and human cardiac preparations. These effects are mediated via A1-adenosine receptors as all effects were sensitive to the selective A1-adenosine receptor antagonist DPCPX. Furthermore, the effects of AP6A on cyclic AMP levels, protein phosphorylation and the L-type calcium current are in accordance with stimulation of A1-adenosine receptors.     

Synthesis and Pharmacological In Vitro Investigations of Novel Shikonin Derivatives with a Special Focus on Cyclopropane Bearing Derivatives

Melanoma is the deadliest form of skin cancer and accounts for about three quarters of all skin cancer deaths. Especially at an advanced stage, its treatment is challenging, and survival rates are very low. In previous studies, we showed that the constituents of the roots of Onosma paniculata as well as a synthetic derivative of the most active constituent showed promising results in metastatic melanoma cell lines. In the current study, we address the question whether we can generate further derivatives with optimized activity by synthesis. Therefore, we prepared 31, mainly novel shikonin derivatives and screened them in different melanoma cell lines (WM9, WM164, and MUG-Mel2 cells) using the XTT viability assay. We identified (R)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-cyclopropyl-2-oxoacetate as a novel derivative with even higher activity. Furthermore, pharmacological investigations including the ApoToxGlo^TM Triplex assay, LDH assay, and cell cycle measurements revealed that this compound induced apoptosis and reduced cells in the G1 phase accompanied by an increase of cells in the G2/M phase. Moreover, it showed hardly any effects on the cell membrane integrity. However, it also exhibited cytotoxicity against non-tumorigenic cells. Nevertheless, in summary, we could show that shikonin derivatives might be promising drug leads in the treatment of melanoma.     

Coaxial Alginate Hydrogels: From Self-Assembled 3D Cellular Constructs to Long-Term Storage

Alginate as a versatile naturally occurring biomaterial has found widespread use in the biomedical field due to its unique features such as biocompatibility and biodegradability. The ability of its semipermeable hydrogels to provide a favourable microenvironment for clinically relevant cells made alginate encapsulation a leading technology for immunoisolation, 3D culture, cryopreservation as well as cell and drug delivery. The aim of this work is the evaluation of structural properties and swelling behaviour of the core-shell capsules for the encapsulation of multipotent stromal cells (MSCs), their 3D culture and cryopreservation using slow freezing. The cells were encapsulated in core-shell capsules using coaxial electrospraying, cultured for 35 days and cryopreserved. Cell viability, metabolic activity and cell–cell interactions were analysed. Cryopreservation of MSCs-laden core-shell capsules was performed according to parameters pre-selected on cell-free capsules. The results suggest that core-shell capsules produced from the low viscosity high-G alginate are superior to high-M ones in terms of stability during in vitro culture, as well as to solid beads in terms of promoting formation of viable self-assembled cellular structures and maintenance of MSCs functionality on a long-term basis. The application of 0.3 M sucrose demonstrated a beneficial effect on the integrity of capsules and viability of formed 3D cell assemblies, as compared to 10% dimethyl sulfoxide (DMSO) alone. The proposed workflow from the preparation of core-shell capsules with self-assembled cellular structures to the cryopreservation appears to be a promising strategy for their off-the-shelf availability.     

Characterization of Oleogels Based on Waxes and Their Hydrolyzates

In this paper, the structuring of liquid oils, also known as oleogelation, is systematically investigated for the first time using a quasi-quaternary mixing system approach. Native waxes with different quantities of wax esters (WE), n-alkanes (hydrocarbons (HC)), fatty acids (FA), and fatty alcohols (FaOH) are applied in mixtures with hydrolyzed waxes to systematically change the composition. Hydrolyzed waxes contain high levels of FA and FaOH. The model systems are investigated on microscopic level (brightfield light microscopy (BFM), cryogenic scanning electron microscopy (cryo-SEM)) as well as on their macroscopic properties (rheology, gel hardness) and calorimetric behavior (differential scanning calorimetry (DSC)). It is found that sunflower wax (SFW)-based gels (12% structurant) become less hard on any admixture. Beeswax (BW)-based gels show significant increases in hardness when 25% and 50% (w/w) hydrolyzate are admixed. This could be related to stepwise crystallization. Further analysis reveals that the dissolution/melting behavior of the wax ester mixtures can be surprisingly well described as ideal solubility of a single pseudocomponent. The approach to unravel the individual contributions of the different species present in waxes is successful and marks a first step to better understand the systematic of wax functionality as oleogelators. Practical Application: The substitution of hardstock fats in structured oil phases is of interest for two reasons. The improved nutritional profile oleogels offer are beneficial for public health while the elimination of palm oil based ingredients appears to be a general public desire. Among the technical solutions for non-TAG oil structuring waxes are very promising. This is primarily due to their availability, prior consumption, potentially low cost for functionality. Currently waxes are technically and scientifically wrongly treated as single components. In order to better utilize the potential of waxes and design future sourcing strategies it is necessary to understand the wax functionality at a compositional/molecular level. This contribution marks the first step into this direction by considering classes of molecules with respect to their contribution to functionality. This understanding is considered as a key for future compositional design.     

In search of trustworthy information: a qualitative study of the search behavior of people with dyslexia in Norway

Universal Access in the Information Society - Access to trustworthy information is a premise for participation in a democratic society. However, locating useful and valid information may not be a...     

Fabrication of multilayer systems combining microfluidic andmicrooptical elements for fluorescence detection

This paper presents the fabrication of a microchemical chip for the detection of fluorescence species in microfluidics. The microfluidic network is wet-etched in a Borofloat 33 (Pyrex) glass wafer and sealed by means of a second wafer. Unlike other similar chemical systems, the detection system is realized with the help of microfabrication techniques and directly deposited on both sides of the microchemical chip. The detection system is composed of the combination of refractive microlens arrays and chromium aperture arrays. The microfluidic channels are 60 μm wide and 25 μm deep. The utilization of elliptical microlens arrays to reduce aberration effects and the integration of an intermediate (between the two bonded wafers) aluminum aperture array are also presented. The elliptical microlenses have a major axis of 400 μm and a minor axis of 350 μm. The circular microlens diameters range from 280 to 300 μm. The apertures deposited on the outer chip surfaces are etched in a 3000-Å-thick chromium layer, whereas the intermediate aperture layer is etched in a 1000-Å-thick aluminum layer. The overall thickness of this microchemical system is less than 1.6 mm. The wet-etching process and new bonding procedures are discussed. Moreover, we present the successful detection of a 10-nM Cy5 solution with a signal-to-noise ratio (SNR) of 21 dB by means of this system