Wetting properties of porous high temperature polymer electrolyte fuel cells materials with phosphoric acid.

Wetting properties of phosphoric acid in porous materials of high temperature fuel cells (HT-PEFC), operating at around 160 °C, are important for cell performance and durability, but the underlying wetting parameters have been unknown so far. Therefore, the influence of phosphoric acid temperature and concentration on the wetting behavior of porous HT-PEFC materials is investigated. The acid filling of gas diffusion and catalyst layers as function of capillary pressure is monitored with X-ray tomographic microscopy under the well defined conditions of an ex situ set-up at temperatures up to 160 °C. For the wetting of gas diffusion layers, with pore sizes in the order of few 10 µm, two opposing trends are shown. With increasing phosphoric acid concentration, less capillary pressure is required, while with increasing temperatures, higher capillary pressures are needed for filling up to a given saturation. The same trends are also found for the contact angle of phosphoric acid on PTFE. A higher contact angle is observed with increasing temperature while increasing the phosphoric acid concentration decreases the contact angle. As both trends are of a similar order of magnitude, the wetting behavior of concentrated (113 wt%) phosphoric acid at 160 °C is astonishingly similar to the wetting behavior of water at room temperature. Another important property for HT-PEFC operation is the filling of cracks in the catalyst layer, which have widths up to 100 µm. For large cracks (>60 µm), a capillary pressure of only 15 mbar was deduced from the measurement, increasing to 30 mbar for cracks between 20 and 60 µm. This, for the first time, allows for assessing the membrane phosphoric acid pressure during fuel cell operation. This can guide the development of improved porous materials for HT-PEFC.

Homogeneity and elemental distribution in self-assembled bimetallic Pd-Pt aerogels prepared by a spontaneous one-step gelation process.

Multi-metallic aerogels have recently emerged as a novel and promising class of unsupported electrocatalyst materials due to their high catalytic activity and improved durability for various electrochemical reactions. Aerogels can be prepared by a spontaneous one-step gelation process, where the chemical co-reduction of metal precursors and the prompt formation of nanochain-containing hydrogels, as a preliminary stage for the preparation of aerogels, take place. However, detailed knowledge about the homogeneity and chemical distribution of these three-dimensional Pd-Pt aerogels at the nano-scale as well as at the macro-scale is still unclear. Therefore, we used a combination of spectroscopic and microscopic techniques to obtain a better insight into the structure and elemental distribution of the various Pd-rich Pd-Pt aerogels prepared by the spontaneous one-step gelation process. Synchrotron-based extended X-ray absorption fine structure (EXAFS) spectroscopy and high-angle annular dark-field (HAADF) scanning transmission electron microscopy (STEM) in combination with energy-dispersive X-ray spectroscopy (EDX) were employed in this work to uncover the structural architecture and chemical composition of the various Pd-rich Pd-Pt aerogels over a broad length range. The Pd80Pt20, Pd60Pt40 and Pd50Pt50 aerogels showed heterogeneity in the chemical distribution of the Pt and Pd atoms inside the macroscopic nanochain-network. The features of mono-metallic clusters were not detected by EXAFS or STEM-EDX, indicating alloyed nanoparticles. However, the local chemical composition of the Pd-Pt alloys strongly varied along the nanochains and thus within a single aerogel. To determine the electrochemically active surface area (ECSA) of the Pd-Pt aerogels for application in electrocatalysis, we used the electrochemical CO stripping method. Due to their high porosity and extended network structure, the resulting values of the ECSA for the Pd-Pt aerogels were higher than that for a commercially available unsupported Pt black catalyst. We show that the Pd-Pt aerogels possess a high utilization of catalytically active centers for electrocatalytic applications based on the nanostructured bimetallic framework. Knowledge about the homogeneity and chemical distribution of the bimetallic aerogels can help to further optimize their preparation by the spontaneous one-step gelation process and to tune their electrocatalytic reactivity.

Quantifying phosphoric acid in high-temperature polymer electrolyte fuel cell components by X-ray tomographic microscopy.

Synchrotron-based X-ray tomographic microscopy is investigated for imaging the local distribution and concentration of phosphoric acid in high-temperature polymer electrolyte fuel cells. Phosphoric acid fills the pores of the macro- and microporous fuel cell components. Its concentration in the fuel cell varies over a wide range (40-100 wt% H3PO4). This renders the quantification and concentration determination challenging. The problem is solved by using propagation-based phase contrast imaging and a referencing method. Fuel cell components with known acid concentrations were used to correlate greyscale values and acid concentrations. Thus calibration curves were established for the gas diffusion layer, catalyst layer and membrane in a non-operating fuel cell. The non-destructive imaging methodology was verified by comparing image-based values for acid content and concentration in the gas diffusion layer with those from chemical analysis.

Dual spectrum neutron radiography: identification of phase transitions between frozen and liquid water.

In this Letter, a new approach to distinguish liquid water and ice based on dual spectrum neutron radiography is presented. The distinction is based on arising differences between the cross section of water and ice in the cold energy range. As a significant portion of the energy spectrum of the ICON beam line at Paul Scherrer Institut is in the thermal energy range, no differences can be observed with the entire beam. Introducing a polycrystalline neutron filter (beryllium) inside the beam, neutrons above its cutoff energy are filtered out and the cold energy region is emphasized. Finally, a contrast of about 1.6% is obtained with our imaging setup between liquid water and ice. Based on this measurement concept, the temporal evolution of the aggregate state of water can be investigated without any prior knowledge of its thickness. Using this technique, we could unambiguously prove the production of supercooled water inside fuel cells with a direct measurement method.

Pt nanoparticles supported on Sb-doped SnO2 porous structures: developments and issues.

In this work, high surface area antimony doped tin oxide (Sb-SnO2) has been synthesized using a modified sol-gel synthesis method. The bulk and surface properties of the metal oxide support have been investigated as a function of the processing conditions. A change in the Sb-SnO2 processing conditions, while preserving an overall invariant bulk composition, led to substantial modification of the surface stoichiometry. Accelerated stability test protocols have shown that the surface composition represents a crucial parameter for the electrochemical stability of Sb-SnO2. Model Pt/Sb-SnO2 electrodes have been developed depositing Pt nanoparticles by magnetron sputtering on the optimized Sb-SnO2 porous surface. A significant enhancement in the corrosion stability upon 1000 potential cycles between 0.5 and 1.5 V (RHE) at 50 mV s(-1) has been observed for the Pt/Sb-SnO2 system compared to Pt/carbon.

Design of polybenzimidazolium membranes for use in vanadium redox flow batteries.

In recent years, polybenzimidazole (PBI) membranes have been proposed for vanadium redox flow batteries (VRFBs) as an alternative to perfluoroalkylsulfonic acid membranes such as Nafion™. Despite their excellent capacity retention, PBI membranes tend to suffer from a low ionic conductivity. The formation of a polybenzimidazolium through an N-alkylation of the benzimidazole core is shown to improve the ionic conductivity of the membrane, with this class of materials having found uses in alkaline fuel cell and water electrolysis systems. However, much less is known about their incorporation into a VRFB. This article describes the use of hexamethyl-p-terphenyl polybenzimidazolium (HMT-PMBI) membranes for a vanadium redox flow battery, with the membrane characteristics in acidic media being related to their performance in a single-cell VRFB setup. A change of the degree of methylation from 56 to 65, 75, and 89% leads to an increase in ionic conductivity, correlated with an increased fraction of free water in the ionomer. The corresponding increase in cell performance is, however, accompanied by a drop in capacity retention. The membrane with a degree of methylation of 65% shows balanced properties, with a 5% higher efficiency and a two times improved capacity retention compared to Nafion™ NR212 over 200 charge-discharge cycles at 200 mA cm-2.

Genetically obese mice: resistance to metastasis of B16 melanoma and enhanced T-lymphocyte mitogenic responses.

The metastasis of B16 melanoma cells differed significantly in obese (ob/ob) and lean (+/?) female mice of strain C57BL/6J. When the mice were inoculated subcutaneously with melanoma cells at 10 to 11 months of age, the primary tumor grew more slowly in obese than in lean littermates and the frequency of lung metastasis was greatly reduced. When the mice were injected with the cells at 4 to 7 months, the primary tumor grew at the same rate in obese and lean mice, but the obese mice again showed a significantly reduced frequency of lung metastasis. That this effect was related to an enhanced immunocompetence in obese mice was supported by the finding that splenic lymphocytes of ob/ob mice showed three times the proliferative response to the T-cell mitogen concanavalin A compared with the proliferative response of lean control mice. The ob/ob mouse may provide a model for the study of enhanced immunocompetence in obese individuals.

Evaluation of analytical markers characterising different drying methods of parsley leaves (Petroselinum crispum L.).

Drying process of parsley leaves from Petroselinum crispum L. can influence the sensory qualities and aromatic taste of this herbal product. Beside oven-dried material, freeze-dried parsley is getting increasingly into the market. In the course of a search for analytical tools to differentiate oven-dried and lyophilised parsley, a HPLC determination of the 6"-O-malonylapiin to apiin ratio was shown to be a suitable marker system. While the ratio is high for fresh and lyophilised leave material, oven-drying leads to demalonylation and, subsequently, to a low malonylapiin--apiin ratio. Additionally, L*a*b colour measurement can be used for quality control to differentiate between different dried parsley raw materials.

Lignans from Linum species of sections Syllinum and Linum.

The aryltetralin lignans 6-methoxypodophyllotoxin, 5'-demethoxy-6-methoxypodophyllotoxin as well as the corresponding 8'-epimers 6-methoxypicropodophyllin, and 5'-demethoxy-6-methoxypicropodophyllin were isolated from suspension cultures of Linum cariense, and 4'-demethyl-6-methoxypodophyllotoxin together with 6-methoxypodophyllotoxin from plants of L. tauricum, which both belong to section Syllinum of the genus Linum. Cell cultures of L. altaicum, L. austriacum ssp. euxinum and L. lewisii belonging to section Linum accumulate the naphthalene lignans justicidin B and isojusticidin B. The different lignans were identified by HPLC and spectroscopic methods.

Comparison of in vivo activation of triamcinolone acetonide- and RU 38486-receptor complexes in the CEM-C7 and IM-9 human leukemic cell lines.

RU 38486 functions as a pure antiglucocorticoid in the human leukemic cell lines CEM-C7 and IM-9. Despite the fact that RU 38486 has been reported to promote considerable in vitro receptor activation to a DNA-binding form, this steroid may be relatively incapable of promoting this physiologically relevant conformational change in vivo. In the experiments reported here the potential ability of RU 38486 to promote in vivo activation has been compared with that of a potent agonist, triamcinolone acetonide. In vivo activation was evaluated by DEAE-cellulose chromatographic analyses of cytosolic extracts prepared from lysed cells which had previously been labeled with either 30 nM [3H]triamcinolone acetonide or [3H]RU 38486 and subsequently incubated at 37 degrees C. Using this anion-exchange procedure, in vivo activation of the agonist-receptor complexes was shown to occur in both a time- and temperature-dependent fashion in both cell lines. This in vivo activation was reflected by progressive decreases in the bound [3H]triamcinolone acetonide associated with the unactivated (high salt-eluting) peaks eluted from DEAE-cellulose, small yet detectable increases in the bound radioactivity eluted in the activated (low-salt eluting) peaks, and a significant increase in the ability of the cytoplasmic [3H]triamcinolone acetonide-receptor complexes to bind to DNA-cellulose. Additional experiments employing DEAE-cellulose chromatography demonstrated that after incubation at 37 degrees C for 1 h, at least some in vivo activation of cytosolic [3H]RU 38486-receptor complexes could be detected in CEM-C7 cells, although the antagonist was less effective than the agonist in facilitating this conformational change. In IM-9 lymphocytes essentially no in vivo activation could be detected using this same protocol. Nuclear translocation assays were also independently performed to evaluate in vivo receptor activation. Incubation of intact cells with 30 nM [3H]triamcinolone acetonide or [3H]RU 38486 for 1 h at 37 degrees C (but not at 0-4 degrees C) revealed that both steroids facilitated translocation in CEM-C7 as well as IM-9 cells, although again the antagonist was less effective than the agonist in this regard. Taken collectively these data indicate that the antagonist RU 38486 is capable of mediating detectable in vivo activation of CEM-C7 cytoplasmic glucocorticoid receptors. These data suggest that complete in vivo stabilization of unactivated receptors by RU 38486 in this cell line may not be sufficient to explain the lack of any agonist activity associated with this synthetic antagonist.(ABSTRACT TRUNCATED AT 400 WORDS)

Non-glucocorticoid receptor-mediated effects of the potent glucocorticoid deacylcortivazol.

Deacylcortivazol (DAC), a potent glucocorticoid, contains a phenyl-pyrazole moiety fused to the 2--3 position of the traditional steroid nucleus. When incubated with glucocorticoid-resistant mutants derived from the glucocorticoid-sensitive human leukemic cell line CEM-C7, DAC caused significant growth inhibition. However, this effect required 1 microM DAC, a concentration 50 times higher than that necessary for glucocorticoid receptor saturation. Cytotoxicity was observed in both mutants containing high-affinity glucocorticoid receptors defective in nuclear translocation and a mutant completely devoid of receptors. Further, in dexamethasone-resistant clones, DAC elicited only marginal increases in the activity of the glucocorticoid-inducible enzyme glutamine synthetase. Clones resistant to high concentrations of DAC could not be directly isolated from CEM-C7. However, stable DAC-resistant clones could be isolated from dexamethasone-resistant subclones of CEM-C7 with a frequency of 1 to 8 x 10(-4). These data are consistent with resistance to DAC being acquired in a two-step process. Our results suggest that the cytotoxicity of DAC at concentrations higher than necessary for glucocorticoid receptor saturation is not mediated by glucocorticoid receptors. Thus, DAC may be a bifunctional compound having both steroid receptor-mediated and receptor-independent cytotoxicity.

A highly flexible electrochemical flow cell designed for the use of model electrode materials on non-conventional substrates.

We present a novel electrochemical flow cell based on a wall-jet configuration to carry out electrochemical investigations under controlled mass transport conditions. The described setup can be applied for investigations similar to those performed with a common rotating disc electrode setup but allows the use of non-conductive and square substrates. This setup thus opens the possibility for the characterization of a new range of materials on a broad range of substrates. Cyclic voltammograms were recorded to assess the cleanliness and good saturation of the cell with inert gas. The performance of the flow cell regarding hydrodynamic experiments was evaluated by probing the oxygen reduction reaction on differently prepared platinum catalysts, including Pt on non-conductive substrates. The high reproducibility of the limiting currents for these samples demonstrates the good functionality, adaptability, and flexibility of the cell.

In vitro activation of rat cardiac glucocorticoid antagonist- versus agonist-receptor complexes.

The synthetic antiglucocorticoid RU 38486 interacts with cardiac cytoplasmic glucocorticoid receptors and competes for in vitro binding with the potent agonist triamcinolone acetonide. In addition to binding to receptors with high affinity, RU 38486 also facilitates the in vitro conformational change in the receptor which is a consequence of the physiologically relevant activation step during which the receptor is converted from a non DNA- to a DNA-binding form. This ability of RU 38486 to promote receptor activation is reflected by both the appropriate shift in the elution profile of [3H]RU 38486-receptor complexes from DEAE-cellulose as well as by an increased binding of these complexes to DNA-cellulose. Although less effective than triamcinolone acetonide, RU 38486 promotes in vitro receptor activation under a variety of experimental conditions, including incubation of labeled cardiac cytosols at 25 degrees C for 30 min or at 15 degrees C for 30 min in the presence of 5 mM pyridoxal 5'-phosphate. Once thermally activated, the cardiac [3H]triamcinolone acetonide and [3H]RU 38486-receptor complexes bind to nonspecific DNA-cellulose with the same relative affinities, as evidenced by the fact that 50% of both activated complexes are eluted at approx. 215-250 mM NaCl. Thus, this pure antiglucocorticoid does promote, at least to some extent, many of the crucial in vitro events including high-affinity binding, activation, and DNA binding which have been shown to be required to elicit a physiological response in vivo.

Homodimer formation is rate-limiting for high affinity DNA binding by glucocorticoid receptor.

The glucocorticoid receptor (GR) is a hormone-inducible transcription factor which activates transcription of specific genes by binding to a DNA sequence present in the promoters of inducible genes. These glucocorticoid response elements (GREs) have a conserved palindromic sequence. Each half-GRE palindrome binds one subunit of GR. We have assessed the relative affinity of GR monomers and homodimers for GRE and determined whether homodimer formation is rate-limiting for high affinity GRE binding. The in vitro affinity of GRE binding by GR homodimers was approximately 2 x 10(-10) M, whereas it was approximately 1 nM for GR monomers. While homodimer:GRE complexes were very stable, monomer:GRE complexes appeared less stable in vitro. At low receptor concentration, GR preferentially bound GRE as a homodimer. Prior dilution of GR (equilibrium shifted to monomers) before addition to a GRE binding reaction resulted in slower kinetics of binding by comparison to parallel reactions in which concentrated (largely homodimeric) GR was added first. Taken together, these experiments suggest that homodimer formation is rate-limiting for high affinity GRE binding. A GRE mutant which contained only a half-binding site and which was unable to bind GR homodimers was also unable to confer glucocorticoid-inducible transcription. Taken together with previous work, these experiments support the model that GR homodimers are required for hormone-dependent activation of transcription and that receptor homodimer formation is rate-limiting for GRE binding.

The alpha-subunit of the epithelial sodium channel is an aldosterone-induced transcript in mammalian collecting ducts, and this transcriptional response is mediated via distinct cis-elements in the 5'-flanking region of the gene.

Aldosterone stimulates Na(+) reabsorption in the collecting ducts by increasing the activity of the epithelial sodium channel, ENaC. Systemic administration of aldosterone increases alpha ENaC mRNA expression in mammalian kidney, suggesting that the alpha ENaC gene is a target for aldosterone action in the distal nephron. To determine whether aldosterone increases alpha ENaC gene transcription, a portion of the alpha ENaC 5'- flanking region coupled to luciferase was transfected into MDCK-C7 cells, a collecting duct cell line with aldosterone-stimulated Na(+) transport. Both dexamethasone and aldosterone stimulated alpha ENaC-coupled reporter gene activity via the glucocorticoid receptor (GR), and this response correlated with the effect of these hormones on endogenous alpha ENaC expression. The aldosterone-stimulated alpha ENaC expression was blocked by actinomycin D, and aldosterone had no effect on alpha ENaC mRNA decay, confirming a transcriptional effect. In HT-29 cells, a GR/mineralocorticoid receptor (MR)-deficient colonic cell line with constitutive alpha ENaC expression, cotransfection with GR or MR restored aldosterone-stimulated alpha ENaC gene transcription, although aldosterone had a functional preference for MR. Analysis of deletion constructs confirmed that a single imperfect glucocorticoid response element (GRE) is necessary and sufficient to confer the aldosterone responsiveness to the alpha ENaC gene promoter in MDCK-C7 and HT-29 cells. These results confirm that alpha ENaC is an aldosterone-induced transcript in the collecting duct and delineates the molecular mechanism for this effect.

In contrast to glucocorticoid receptors, mineralocorticoid receptors are not autoregulated in rat distal colon epithelia.

Glucocorticoids and mineralocorticoids exert profound effects on electrolyte balance in the rat distal colon by binding with high affinity and specificity to intracellular receptor proteins, termed glucocorticoid (GR) and mineralocorticoid (MR) receptors, respectively. Hormonal regulation of GR and MR expression represents an important mechanism for maintaining appropriate tissue sensitivity to these two classes of adrenocorticosteroids. In the present study the corticosteroid regulation of the expression of these two rat colonic receptors has been evaluated at the protein and mRNA levels. Preliminary experiments demonstrated that colonic MR and GR binding levels are significantly increased (approximately 60%) after adrenalectomy (14 days). Experiments were therefore performed to test the hypothesis that these increases in binding levels correlate with increased GR and MR mRNA and protein levels. Receptor mRNA levels were quantitated via ribonuclease protection assays using [32P]cRNA probes specific for either GR or MR mRNA, and GR and MR protein levels were quantitated via Western immunoblotting using the anti-GR BuGR2 and anti-MR hMRsN antibodies. Fourteen days after adrenalectomy, significant increases in GR mRNA (2.1-fold) and protein levels (1.6-fold) were detected. In contrast, neither MR mRNA nor protein levels were up-regulated by removal of endogenous corticosteroids. Additionally, GR and MR mRNA levels were measured after ip injection of adrenalectomized rats with pharmacological doses of either the pure glucocorticoid agonist RU 28362 or the mineralocorticoid agonist aldosterone in combination with the glucocorticoid antagonist RU 38486 (blocks aldosterone binding to the GR). Multiple ip injections of RU 28362 resulted in a significant decrease (80%) in GR mRNA levels without affecting MR mRNA levels. In contrast, multiple ip injections of aldosterone (plus RU 38486) had no effect on either MR or GR mRNA levels. A single ip injection of RU 28362 resulted in a detectable decrease (30%) in GR mRNA levels and again had no effect on MR mRNA levels. Although a single ip injection of aldosterone did not down-regulate MR mRNA levels, it did elicit a significant decrease (45%) in GR mRNA levels. Taken collectively, these data demonstrate that rat colonic GR and MR mRNA and protein levels are differentially up-regulated after removal of endogenous corticosteroids. Additionally, these data demonstrate that in response to its cognate ligand, the GR is capable of homologously down-regulating its own mRNA and protein levels. However, in response to its cognate ligand, the MR appears incapable of homologously down-regulating its own mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of 1,10-phenanthroline on the binding of activated rat hepatic glucocorticoid-receptor complexes to deoxyribonucleic acid-cellulose.

1,10-Phenanthroline, a metal ion chelator, inhibits the binding of previously activated (25 C for 30 min) rat hepatic [3H]triamcinolone acetonide (3H-labeled 9-fluoro-11 beta, 21-dihydroxy-16 alpha, 17-1-[1-metylethylidenebis(oxy)]pregna-1,4-diene-3,20-dione ([3H]TA)-receptor complexes to DNA-cellulose. The observed inhibition increases as the temperature of the preincubation with chelator is increased from 0 to 25 C. Fifty percent of the maximal inhibition (greater than 90%) detected at 25 C is achieved with 1 mM 1,10-phenanthroline. The observed inhibition is not the consequence of DNA degradation by 1,10-phenanthroline-Cu2+ complexes, since preincubation of activated cytosol with neocuproine (2,9-dimethyl-1,10-phenanthroline), a potent Cu2+ chelator, fails to block the subsequent inhibition of DNA-cellulose binding by 1,10-phenanthroline. The failure of other chelators which complex siilar metal ions (alpha, alpha'-dipyridyl,8-hydroxyquinoline, 2,2',2"-tripyridine, EDTA, EGTA, and Na azide) to inhibit DNA-cellulose binding suggests that the effectiveness of 1,10-phenanthroline does not result from removal of a required free metal ion(s) but, rather, from a specific interaction with a metal ion(s) which may be located within the activated receptor protein. The observed inhibition is dependent on the metal chelating properties of 1,10-phenanthroline, since preincubation with several divalent metal cations (Zn2+, Co2+, and Ni2+) which are known to be chelated by this compound block its subsequent inhibitory effect. Ferroin (1,10-phenanthroline-ferrous sulfate complex) and 1,7-phenanthroline (nonchelating isomer) also fail to inhibit DNA-cellulose binding. The inhibition mediated by 1,10-phenanthroline persists after gel filtration, suggesting that 1,10-phenanthroline associated with a macromolecule is the effective form of the inhibitor, rather than free 1,10-phenanthroline. Finally, 1,10-phenanthroline appears to interact directly with activated [3H]TA-receptor complexes, since it alters their net charge and results in their elution from DEAE-cellulose at a salt concentration characteristic of unactivated complexes. Collectively, the data suggest that the activated [3H]TA-receptor complex is a metalloprotein and that the metal ion(s) may be associated directly with the DNA-binding site or may regulate this site indirectly through an allostreic mechanism.

Use of [3H]cortivazol to characterize glucocorticoid receptors in a dexamethasone-resistant human leukemic cell line.

ICR 27 is a mutant derived from the glucocorticoid-sensitive human leukemic cell line CEM C7 that has been characterized as glucocorticoid receptor negative based on its ability to specifically bind [3H]dexamethasone ([3H]DEX). We used the pyrazolosteroid [3H]cortivazol ([3H]CVZ) to determine whether ICR 27 cells actually contain glucocorticoid receptors and, if so, whether these receptors can mediate physiological effects. Scatchard analysis of the binding of [3H]CVZ to cytosol from ICR 27 cells was consistent with a single class of receptors of uniform affinity (0.7 nM). Cytosolic [3H]CVZ complexes had a sedimentation coefficient of 4.6S on linear sucrose gradients and eluted from DEAE-cellulose columns at a potassium phosphate concentration of 250 mM. CVZ also competed with [3H]DEX mesylate for binding to a 96,000 mol wt protein. Incubation of ICR 27 cells with CVZ caused 50% growth inhibition and 50% maximal induction of glutamine synthetase activity at concentrations of 20 and 35 nM, respectively. Elution profiles of [3H]CVZ complexes from DEAE-cellulose columns showed that complexes formed upon thermal activation were relatively unstable, and little or no increase in binding of [3H]CVZ-receptor complexes to DNA-cellulose was observed. Thus, [3H]CVZ identifies functional glucocorticoid receptors in a cell line previously described as DEX resistant. Although the binding of [3H]CVZ to activated receptors in vitro appears unstable, high concentrations of CVZ may facilitate stabilization of activated complexes that can mediate both anabolic and catabolic effects.

Inhibitory effects of helenalin and related compounds on 5-lipoxygenase and leukotriene C(4) synthase in human blood cells.

The sesquiterpene lactone helenalin, which can be isolated from several plant species of the Asteraceae family, is a potent anti-inflammatory and antineoplastic agent. In agreement, alcohol extracts of these plants are used for local external treatment of inflammatory conditions. Since leukotrienes are important mediators in inflammatory processes, the inhibitory effects of helenalin and some derivatives on leukotriene (LT) biosynthesis were studied. Treatment of human platelets with helenalin provoked irreversible inhibition of LTC(4) synthase in a concentration- and time-dependent manner with an IC(50) of 12 microM after a 60 min preincubation. 11alpha,13-Dihydrohelenalin acetate was less potent. Interestingly, individual donors could be divided into two distinct groups with respect to the efficacy of helenalin to suppress platelet LTC(4) synthase. In human granulocytes, helenalin inhibited both the 5-lipoxygenase (IC(50) 9 microM after 60 min preincubation) and LTC(4) synthase in a concentration- and time-dependent fashion. In contrast, the drug was without effect on LTA(4) hydrolase. The GSH-containing adducts (2beta-(S-glutathionyl)-2,3-dihydrohelenalin and 2beta-(S-glutathionyl)-2,3,11alpha,13-tetra hydrohelenalin acetate) did not significantly inhibit LTC(4) synthase. The present results indicate a mechanism for the anti-inflammatory effect of helenalin and related compounds.

Agonist-specific modulation of glucocorticoid receptor-mediated transcription by immunosuppressants.

Although the immunosuppressive drugs FK506, rapamycin and cyclosporin A have been reported to potentiate transcriptional activation mediated by a non-saturating concentration of the glucocorticoid receptor agonist dexamethasone, the precise mechanism(s) underlying these responses remains unclear. The murine L-929-derived LMCAT cell line stably transfected with the mouse mammary tumor virus promoter-chloramphenicol acetyl transferase reporter gene construct was utilized in the present study to further investigate the mechanism(s) underlying this dexamethasone potentiation as well as the possible agonist specificity of this potentiation. The present data demonstrate that pretreatment (2 h) of LMCAT cells with 10 microM FK506, rapamycin or cyclosporin A results in the potentiation of reporter gene transcription mediated not only by dexamethasone (approximately 12-fold), but also by hydrocortisone (approximately 6-fold) and triamcinolone acetonide (approximately 2.5-fold). In sharp contrast, the data show for the first time that pretreatment with any one of these immunosuppressive drugs suppresses (approximately 2-8-fold) the transcriptional responses mediated by corticosterone, deoxycorticosterone, and cortexolone. Pretreatment of intact LMCAT cells with FK506 increases the subsequent whole cell specific binding of [3H]dexamethasone, but does not increase specific cytoplasmic binding when the tritiated agonist is added directly to cytosolic extracts prepared from the pretreated cells. These data suggest that the FK506-mediated potentiation of the transcriptional responses induced by some agonists, like dexamethasone, may be related to the ability of this immunosuppressant to inhibit the membrane-associated multidrug resistance (MDR) P-glycoprotein, which actively extrudes some steroids from cells. Identical pretreatment with FK506 has no detectable effect on the subsequent whole cell specific binding of [3H]corticosterone, a steroid which is not effectively extruded by the MDR pump. Two additional MDR pump inhibitors, verapamil and quinidine, potentiate (30-fold) the dexamethasone-mediated transcriptional response as expected, but have no detectable effects on a corticosterone-mediated transcriptional response. Unlike immunosuppressive drugs, these ion channel blockers do not bind to receptor-associated immunophilins (FK506-binding proteins or cyclophilins). Collectively, these results suggest that immunosuppressants potentiate a dexamethasone-mediated transcriptional response in LMCAT cells by inhibiting efflux of this steroid. In contrast, these drugs appear to suppress a corticosterone-mediated transcriptional response by a different mechanism, perhaps one involving their binding to glucocorticoid receptor-associated immunophilins.