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The kinetics of the chemical vapor deposition (CVD) of graphene on Cu in CH4 + H2 were investigated by monitoring the graphene flake size as a function of CVD growth time. A growth model was set up which relates the CVD parameters to the mass action constant Qexp of the methane decomposition reaction toward graphene at a given temperature T. Graphene growth was shown to proceed from pre-equilibrated adsorbed carbon (Cad) within a wide CVD parameter range. The model not only leads to the correct scaling relation of the growth kinetics but quantitatively determines how far the CVD parameters deviate from thermal equilibrium and correctly predicts the absolute flake size increase per time. Fitting experimental data delivers the energy barrier for carbon detachment from the graphene island edge (Edet = 4.7 ± 0.3 eV) and the methane decomposition entropy toward Cad on Cu (ΔdecS° = 260 ± 20 J mol-1 K-1). The latter value is used to estimate the vanishingly small Cad equilibrium concentration of 3 × 10-10 monolayers at 1045 °C. The universal validity of the model is proven by comparison with literature data providing the correct order of magnitude growth velocities up to 1000 μm/h. The performed reactor experiments deliver data that match the predicted flake growth velocity with a precision of about 50%. The obtained results can be used to calibrate any hot wall CVD reactor setup for the methane decomposition reaction toward graphene on Cu. The description can be directly applied for any hydrocarbon in the gas feed, and the technique can be easily applied for other catalytic support surfaces.

We outline our achievements in developing electron transparent, leak-tight membranes required for environmental photoelectron spectroscopy (PES). We discuss the mechanical constraints limiting the achievable membrane size and review the development of growth protocols for the chemical vapor deposition (CVD) of single-crystalline graphene on highly (111) textured Cu foils serving as membrane material. During CVD growth, Cu tends to develop a mesoscopic staircase morphology consisting of alternating inclined surface planes, irrespective of whether the covering graphene film or the substrate are single-crystalline. This morphology remains imprinted even when converting the film into freestanding graphene, which affects its mechanical properties. Determining the number of carbon layers in freestanding graphene, we show that membranes reported to suspend over distances larger than 20 µm most likely consist of few-layer graphene. The Raman band signature often used to confirm monolayer graphene rather relates to graphene with turbostratic stacking. The vertical corrugation of freestanding graphene was shown to be almost absent for tri- and four-layer-thick graphene but substantial for bilayer and especially for monolayer graphene. The corrugation is reduced when mechanically straining the freestanding graphene through thermal expansion of the supporting frame, especially flattening membrane areas with imprinted staircase morphology. The electron signal attenuation through supported and freestanding graphene was determined as a function of the electron kinetic energy, verifying that large-area graphene-based electron windows have sufficient electron transparency required for environmental PES. Meanwhile, we managed to cover 100 µm-sized single holes by few-layer graphene up to a coverage fraction of over 99.9998%, as deduced when applying 10 mbar air on one side of the sealing membrane without detecting any measurable pressure increase on its ultrahigh vacuum side. The reported achievements will pave the way toward the development of laboratory-based environmental PES.

Atorvastatin calcium (Lipitor®, Sortis®) is a well-established cholesterol synthesis enzyme (CSE) inhibitor commonly used in the therapy of hypercholesterolemia. This drug is known to be sensitive to acid treatment, but only little data has been published on the structures of the degradation products. Here we report the identification of two novel degradation products of atorvastatin, which are formed only under drastic acidic conditions. While treatment with conc. sulfuric acid led to a loss of the carboxanilide residue (accompanied by an expectable lactonization/dehydration process in the side chain), treatment with conc. aqueous hydrochloric acid gave a complex, bridged molecule under C–C-bond formation of the lactone moiety with the pyrrole, migration of the isopropyl group and loss of the carboxanilide residue. The novel degradation products were characterized by NMR spectroscopy, HRMS data and X-ray crystal structure analysis.

Background Survival in recurrent ependymomas in children and adolescents mainly depends on the extent of resection. Studies on repeated radiotherapy and chemotherapy at relapse have shown conflicting results. Methods Using data from the German multi-center E-HIT-REZ-2005 study, we examined the role of local therapy and the efficacy of chemotherapy with blockwise temozolomide (TMZ) in children and adolescents with recurrent ependymomas. Results Fifty-three patients with a median age of 6.9 years (1.25–25.4) at first recurrence and a median follow-up time of 36 months (2–115) were recruited. Gross- and near-total resection (GTR/NTR) were achieved in 34 (64.2%) patients and associated with a markedly improved 5-year overall survival (OS) of 48.7% vs. 5.3% in less than GTR/NTR. Radiotherapy showed no improvement in OS following complete resection (OS: 70 (CI: 19.9–120.1) vs. 95 (CI: 20.7–169.4) months), but an advantage was found in less than GTR/NTR (OS: 22 (CI: 12.7–31.3) vs. 7 (CI: 0–15.8) months). Following the application of TMZ, disease progression was observed in most evaluable cases (18/21). A subsequent change to oral etoposide and trofosfamide showed no improved response. PF-A EPN were most abundant in relapses (n = 27). RELA-positive EPN (n = 5) had a 5-year OS of 0%. Conclusion The extent of resection is the most important predictor of survival at relapse. Focal re-irradiation is a useful approach if complete resection cannot be achieved, but no additional benefit was seen after GTR/NTR. Longer-term disease stabilization (>6 months) mediated by TMZ occurred in a small number of cases (14.3%).

The efficiency of photoelectrochemical tandem cells is still limited by the availability of stable low band gap electrodes. In this work, we report a photocathode based on lithium doped copper(II) oxide, a black p-type semiconductor. Density functional theory calculations with a Hubbard U term show that low concentrations of Li (Li0.03Cu0.97O) lead to an upward shift of the valence band maximum that crosses the Fermi level and results in a p-type semiconductor. Therefore, Li doping emerged as a suitable approach to manipulate the electronic structure of copper oxide based photocathodes. As this material class suffers from instability in water under operating conditions, the recorded photocurrents are repeatedly misinterpreted as hydrogen evolution evidence. We investigated the photocorrosion behavior of LixCu1−xO cathodes in detail and give the first mechanistic study of the fundamental physical process. The reduced copper oxide species were localized by electron energy loss spectroscopy mapping. Cu2O grows as distinct crystallites on the surface of LixCu1−xO instead of forming a dense layer. Additionally, there is no obvious Cu2O gradient inside the films, as Cu2O seems to form on all LixCu1−xO nanocrystals exposed to water. The application of a thin Ti0.8Nb0.2Ox coating by atomic layer deposition and the deposition of a platinum co-catalyst increased the stability of LixCu1−xO against decomposition. These devices showed a stable hydrogen evolution for 15 minutes.

We demonstrate the remote excitation and detection of surface-enhanced Raman scattering (SERS) from graphene using a silver nanowire as a plasmonic waveguide. By investigating a nanowire touching a graphene sheet at only one terminal, we first show the remote excitation of SERS from graphene by propagating surface plasmon polaritons (SPPs) launched by a focused laser over distances on the order of 10 μm. Remote detection of SERS is then demonstrated for the same nanowire by detecting light emission at the distal end of the nanowire that was launched by graphene Raman scattering and carried to the end of the nanowire by SPPs. We then show that the transfer of the excitation and Raman scattered light along the nanowire can also be visualized through spectrally selective back focal plane imaging. Back focal plane images detected upon focused laser excitation at one of the nanowire's tips reveal propagating surface plasmon polaritons at the laser energy and at the energies of the most prominent Raman bands of graphene. With this approach the identification of remote excitation and detection of SERS for nanowires completely covering the Raman scatterer is achieved, which is typically not possible by direct imaging.

The aliphatic heterocycles piperidine and morpholine are core structures of well-known antifungals such as fenpropidin and fenpropimorph, commonly used as agrofungicides, and the related morpholine amorolfine is approved for the treatment of dermal mycoses in humans. Inspired by these lead structures, we describe here the synthesis and biological evaluation of 4-aminopiperidines as a novel chemotype of antifungals with remarkable antifungal activity. A library of more than 30 4-aminopiperidines was synthesized, starting from N-substituted 4-piperidone derivatives by reductive amination with appropriate amines using sodium triacetoxyborohydride. Antifungal activity was determined on the model strain Yarrowia lipolytica, and some compounds showed interesting growth-inhibiting activity. These compounds were tested on 20 clinically relevant fungal isolates (Aspergillus spp., Candida spp., Mucormycetes) by standardized microbroth dilution assays. Two of the six compounds, 1-benzyl-N-dodecylpiperidin-4-amine and N-dodecyl-1-phenethylpiperidin-4-amine, were identified as promising candidates for further development based on their in vitro antifungal activity against Candida spp. and Aspergillus spp. Antifungal activity was determined for 18 Aspergillus spp. and 19 Candida spp., and their impact on ergosterol and cholesterol biosynthesis was determined. Toxicity was determined on HL-60, HUVEC, and MCF10A cells, and in the alternative in vivo model Galleria mellonella. Analysis of sterol patterns after incubation gave valuable insights into the putative molecular mechanism of action, indicating inhibition of the enzymes sterol C14-reductase and sterol C8-isomerase in fungal ergosterol biosynthesis.

Resveratrol as well as caloric restriction were shown to extend lifespan in some model organisms and may possibly delay onset of ageing-related diseases in humans. Yet, resveratrol supplementation does not always extend lifespan of animal models or improve health status of humans. Because of interindividual differences in human microbiota, resveratrol metabolite production in the gut differs. While some individuals produce lunularin and dihydroresveratrol in their gut, others produce dihydroresveratrol only. Therefore, we addressed the question whether these metabolites differ in their biological impact on ageing and intraperitoneally injected 13-month-old C57BL/6JRj mice on an ad-libitum (AL) HFD with resveratrol, dihydroresveratrol or lunularin (24 mg/kg bodyweight; 3 times/week). Compared to mice injected with vehicle (AL-control), resveratrol and dihydroresveratrol did not change bodyweight and had no impact on insulin or glucose levels while lunularin slightly reduced feed intake and bodyweight gain. CR-mice showed lowered cholesterol, insulin and leptin levels, elevated adiponectin and phosphorylated AMPK levels in liver as well as increased transcription of Pck1 and Pgc1α when compared to the AL-control. In contrast, injections with the test substances did not change these parameters. We therefore conclude that in our model, resveratrol, lunularin and dihydroresveratrol did not act as CR mimetics.

The present study analyses the exclusive use of absorbable suture material (Vicryl(®), Ethicon, Germany) in the fixation of transposed bone segments in craniofacial surgery without modification of the osteotomy design. Among 129 children up to 24 months of age, osteosynthesis was conducted exclusively with Vicryl(®) sutures. The stability of postoperative results was evaluated and possible foreign body reactions were examined within the framework of clinical and radiological routine checks. All examined children exhibited stable postoperative conditions while the length of hospital stay was not affected. X-ray examinations of the skull in two planes demonstrated good bony union in all cases. Relevant foreign body reactions were not observed. The exclusive application of absorbable suture material enables stable and cost effective osteosynthesis. Significant foreign body reactions were not observed. The exclusive use of absorbable sutures did not alter the osteotomy design.

The oxidative carbon removal from Cu foils was systematically studied together with the resulting suppression of the graphene flake nucleation during subsequent chemical vapor deposition (CVD). The carbon content was determined by segregating bulk dissolved carbon during a special rapid cooling process towards the surface, where it was detected by x-ray photoelectron spectroscopy. After exposure to a standardized hydrogen pretreatment, the Cu foils deliver irregularly distributed CVD grown flakes with diameters of 0.1–20 μm. Such foils still contain 40–240 monolayers of carbon, which could be removed by exposure to an oxygen partial pressure of ∼10−5 mbar diluted in 1 mbar Ar carrier gas. This controlled procedure allowed the quantification of the carbon removal while inducing exclusively reversible restructuring of the Cu foil. Only when lowering the carbon content to values well below 1 monolayer per entire Cu foil, the flake nucleation during CVD drops to values below 1 flake/mm2. We also show that it is the absence of carbon and not the presence of oxygen which accounts for the low flake density and that graphene can be grown with up to mm sized domains. Our data question many proposed explanations why certain growth protocols deliver graphene of comparable high quality.

Atypical teratoid/rhabdoid tumors (AT/RTs) are commonly regarded as immunologically cold tumors, as they rank among malignancies with the lowest mutational load. However, low mutational burden is not necessarily correlated with poor immunogenicity, as tumor-exclusive peptides bound in the tumor cell’s MHC represent potential targets for immune responses. Indeed, in a first set of n=17 AT/RT samples we could determine by immunohistochemistry that 1.8% of all cells within the tumors were T-cells. MHC class I, class II, and PD-L1 was expressed on 12.9%, 3%, and 5.1% of the tumor cells, respectively. To investigate whether the ligandome of AT/RTs contained tumor-exclusive peptides we then analyzed 23 centrally reviewed AT/RTs using LC-MS and subsequent bioinformatical validation. In 14 (61%) and 21 (91%) of the 23 patients, tumor-exclusive peptides were identified (59 MHC class I and 153 class II peptides in total). Of these, 47/109 peptides were exclusive for one tumor, demonstrating the high individuality of the AT/RT ligandome. Representative examples of these ligandome peptides proved to be immunogenic in T-cell priming assays. Mutation analysis revealed a median of 11 (range 1-166) tumor-specific mutations dispersed over the entire genome. In silico MHC-binding prediction showed that these neoantigenic peptides had a similar binding affinity compared to their ligandome counterparts. In conclusion, tumor-exclusive ligandome peptides can be identified in the vast majority of AT/RT patients. In silico and in vitro analyses qualify them as suitable candidates for personalized cancer vaccines.

The space-based gravitational wave detector LISA (Laser Interferometer Space Antenna) requires a high performance position sensor in order to measure the translation and tilt of the free flying test mass with respect to the LISA optical bench. Here, we present a mechanically highly stable and compact setup of a heterodyne interferometer combined with differential wavefront sensing for the tilt measurement which serves as a demonstrator for an optical readout of the LISA test mass position. First results show noise levels below 1 nm/√Hz and 1 μrad/√Hz, respectively, for frequencies < 10−3 Hz.

A series of novel N-alkyl tetra- and perhydroquinoline derivatives and their hydrochlorides were prepared from tetrahydro- or trans-perhydroquinoline by direct alkylation with alkyl halides and subsequent precipitation with HCl gas. The antimicrobial activity of the resulting amines was evaluated in an agar diffusion assay. The minimal inhibitory concentrations (MIC) of the active com-pounds were determined by the microdilution method. In contrast to the tetra-hydroquinolines, the perhydro analogues showed significant antifungal activity. In an assay for the detection of target enzymes in ergosterol biosynthesis, N-undecylperhydroquinoline was identified as an inhibitor of Δ8,7-isomerase.

Opioids potently inhibit a number of physiological and pathophysiological effects such as pain and inflammation in the brain and the periphery. One of the targets of opioids mediating such effects is the proinflammatory transcription factor NF-κB. In neuronal cells, opioids inhibit this factor by inducing I-κB independently on calcium, involving the opioid-mediated activation of the transcription factor AP-1. However, when and how precisely NF-κB is modulated by opioids in T cells are unknown. By using the TNF-triggered, NF-κB–mediated induction of IL-8 mRNA in primary human T cells and Jurkat T cells, in this study we show that opioids inhibit NF-κB in T cells as well, but that the underlying mechanisms are different from those observed in neuronal cells. We found that stimulation of the T cells with opioids resulted in a significant inhibition of the TNF-triggered ubiquitination and degradation of I-κB. Additionally, an opioid-mediated induction of the deubiquitinating enzyme ubiquitin-specific protease 15 was observed, which is known to inhibit the NF-κB pathway by stabilizing I-κB. The induction of ubiquitin-specific protease 15 was dependent on calcium and the transcription factor NFAT. Activation of AP-1 and induction of I-κB in response to the opioids were not observed in the T cells. These results indicate that μ opioid receptors, which mediate the effects in both cell types, might be coupled to different effector cascades in the different cell types, which may then result in cell type–specific effects of the drugs.

We investigated the structure and crystalline quality of monolayer graphene grown by hydrogen and methane chemical vapor deposition (CVD) on polycrystalline Cu foils. Our data show that the high temperature hydrogen pretreatment of the Cu foil has to be performed at a sufficiently high H 2 pressure in order to avoid graphene (g) formation already during the pretreatment, which limits the achievable domain size during subsequent growth in the CH 4 /H 2 mixture. Methane–hydrogen CVD sustains g growth but induces the faceting of the Cu substrate. Characterization by low energy electron microscopy evidenced a staircase Cu substrate morphology of alternating (4 1 0) and (1 0 0) planes interrupted by (n 1 1) type facets. The g flakes cover the staircase shaped support as a coherent layer. The polycrystalline film mostly contains rotational domains that are preferentially, but not strictly, aligned with respect to the stepped support surface. The substrate induced corrugated morphology occurs also underneath large single crystalline flakes and is transferred to suspended membranes, produced by etching the Cu underneath the graphene. Thus, membranes manufactured from g-Cu are non flat. This explains their reported softened elastic response and the formation of so called nanorippled graphene after transfer from the Cu support which deteriorates its electrical conductivity.

Opioids are irreplaceable for the treatment of severe pain. However, opioid-induced immunomodulation affects therapies. Here we report that treatment of human T lymphocytes with the opioids fentanyl, methadone, loperamide and beta-endorphin resulted in a strong induction of the cytokine interleukin-4. In contrast, morphine and buprenorphine induced markedly and significantly lower levels of interleukin-4 mRNA and protein. These findings suggest agonist-biased μ opioid receptor signaling in T cells. In the future, better knowledge about agonist-specific immunomodulatory effects of opioids offers the possibility to select drugs for a therapy with more favorable and/or less detrimental side effects in immune cells.

Stereophotogrammetry enables a simple and radiation free longitudinal analysis of skull asymmetries: in a three-dimensional coordinate system various distances (length, breadth, cephalic index, oblique diameters, ear shift, head circumference) can be analyzed. We also defined separate volume sections in order to further quantify the degree of asymmetry in the posterior and anterior components of both sides of the head. In 51 infants (mean age, 6 months; SD 0.97) with positional plagiocephaly, we determined these parameters at the beginning as well as at the end of molding helmet therapy (mean therapy time 4.9 months). Thirty-seven infants without positional deformity (mean age, 6.4 months; SD 0.3) served as control group and provided data about what appears to be normal and how these parameters change during growth over a comparable period of time. Compared with the control group, the plagiocephalic heads were more brachycephalic, but closely approximated the normal shape under molding therapy. The striking volume difference between the left and right posterior sections in the plagiocephalic children (the mean volume of the flattened side being 21 % smaller than the one on the contralateral side) improved as well (to a residual difference of mean 8 %) and ended up with a value close to the control group (mean 6 %). There is a broad clinical application area for stereophotogrammetry analyzing skull morphology: In plagiocephalic infants we demonstrate impressive changes of head shape under molding therapy; in normal-looking infants we describe the extent of unperceived asymmetry.

Opioids and cannabinoids modulate T lymphocyte functions. Many effects of the drugs are mediated by μ-opioid receptor and cannabinoid receptor type 1 (CB1), respectively. These two receptors are strikingly similar with respect to their expression in T cells and the mechanisms by which they mediate modulation of T cell activity. Thus, μ-opioid receptors and CB1 not expressed in resting primary human and Jurkat T cells. However, in response to the cytokine IL-4, the epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A, and activation of T cells, functional μ-opioid receptors and CB1 are induced. The induced receptors mediate inhibition of T cell signaling and, thereby, IL-2 production, a hallmark of activated T cells. Although coupled to inhibitory G proteins, μ-opioid receptors and CB1 produce a remarkable increase in cAMP levels in T cells stimulated with opioids and cannabinoids, which is a key mechanism for the inhibition of T cell signaling.

Opioids potently modulate neuronal functions, for example, by regulating the activity of transcription factors. Here, we investigated the effect of morphine on the activity of the transcription factor nuclear factor κB (NF-κB). Establishing cellular models for our investigations, we demonstrated that NF-κB mediated the tumor necrosis factor (TNF)-induced transcription of the cannabinoid receptor type 1 gene in primary fetal striatal neurons from rats and the human neuroblastoma cell line SH SY5Y. The activity of NF-κB in these models was strongly inhibited by morphine, which was achieved by a marked up-regulation of the inhibitor of nuclear factor-κB (IκB). The opioid-induced up-regulation of IκB was dependent on the transcription factors NF-κB itself and activator protein-1 (AP-1). In fact, stimulation of the cells with morphine resulted in a transient activation of NF-κB and a strong induction of c-Fos, one of the constituents of AP-1. This resulted in IκB levels significantly exceeding the basal, constitutive levels of IκB. These data, together with experiments in which AP-1 and IκB were down-regulated by decoy oligonucleotides and siRNA, suggest that the morphine-induced activation of AP-1 and the subsequent overexpression of IκB are key factors in the inhibition of NF-κB by the drug. In contrast, stimulation of primary neurons from rats and SH SY5Y cells with TNF, which is a classic activator of NF-κB, resulted in a resynthesis of IκB, in which the basal levels of IκB were restored only but did not result in an activation of AP-1 and overexpression of IκB.

Objective: The aim of this study was to investigate the effect of the epigenetic modifiers trichostatin A and 5-aza-2’-deoxycytidine on the expression of the cannabinoid receptors CB1 and CB2 and µ-opioid receptors in human SH SY5Y neuroblastoma cells and human Jurkat T lymphocytes. Methods: Using quantitative real-time RT-PCR, mRNA specific for the aforementioned receptors was determined. The functionality of the induced receptors was determined by analyzing the effect of the ligands to regulate intracellular cAMP. Results: We demonstrated that treatment of SH SY5Y cells, which endogenously express µ-opioid receptors and CB1, but not CB2, resulted in de novo induction of CB2, while mRNA levels of CB1 and µ-opioid receptors were not significantly altered. In contrast, treatment of Jurkat lymphocytes, which endogenously express CB2, but not CB1 and µ-opioid receptors, resulted in de novo induction of CB1 and µ-opioid receptors, while mRNA levels of CB2 were not significantly altered. Furthermore, the functionality of the induced µ-opioid receptors and CB1 in the Jurkat cells was demonstrated. Conclusions: Our data suggest an epigenetically regulated expression of cannabinoid receptors and µ-opioid receptors. Their induction by epigenetic modifiers in distinct cells of the nervous and immune system might result in increased effects of the cognate drugs on neuronal and immune functions. Such modifications might be useful for novel therapies for various disorders, e.g. multiple sclerosis, where the elevated transmission of cannabinoid or opioid signals is beneficial.

Pharmacokinetic enhancers (boosters) are compounds used in combination with a primary therapeutic agent (drug) and are not used for their direct effects on the disease but because they enhance or restore the activity of the primary agent. Hence, in certain cases, they represent an indispensable escort for enzyme-labile drugs. Pharmacokinetic enhancers can exert their activity on different ways. In the most common case, they inhibit enzymes such as human cytochrome P450 enzymes in the liver or other organs and, thereby, block or reduce undesired metabolism and inactivation of the primary drug. In this review, an overview will be given on the therapeutically most important classes of pharmacokinetic enhancers like β-lactamase inhibitors, inhibitors of CYP (cytochrome P450) enzymes in HIV therapy and hepatitis C, boosters for fluoropyrimidine-type anticancer agents, compounds utilized for enabling therapy of Parkinson’s disease with levodopa, and others. Inhibitors of efflux pumps in both pathogenic bacteria and tumor cells will be addresses shortly.

Opioids are widely used for the treatment of severe pain. However, it is also known that opioids, in particular morphine, cause immunosuppression. Therefore, their use may complicate treatment of persons with an already impaired immune system, e.g., patients suffering from cancer or AIDS. We investigated the mechanisms of opioid-induced immunosuppression in primary human T lymphocytes and the human T cell line Jurkat. We demonstrated that morphine and the endogenous opioid β-endorphin inhibited the transcription of IL-2 in activated human T lymphocytes as well as the activation of the transcription factors AP-1, NFAT, and NF-κB, which transactivate IL-2. In addition, the TCR-induced calcium flux and MAPK activation were inhibited by the opioids, as well as proximal signaling events, such as the phosphorylation of the linker for activation of T cells and Zap70. A more detailed characterization of the mechanism revealed that incubation of T cells with the opioids caused a marked increase in cAMP. This in turn activated protein kinase A, which augmented the kinase activity of C-terminal Src kinase bound to phosphoprotein associated with glycosphingolipid-enrich microdomains, resulting in a further enhancement of the tonic inhibition of the leukocyte-specific protein tyrosine kinase Lck, thereby blocking the initiation of TCR signaling. These effects were mediated by μ opioid receptors. Our findings contribute to the understanding of immunosuppressive side effects of morphine. Since β-endorphin is expressed and secreted by immune effector cells, including T cells, and up-regulated in these cells by various stimuli, our data also suggest an inhibitory role for β-endorphin in the physiological regulation of T cell activation.

We have recently shown that the activation of the rat mu-opioid receptor (MOPr, also termed MOR1) by the mu-agonist [D-Ala(2), Me Phe(4), Glyol(5)]enkephalin (DAMGO) leads to an increase in phospholipase D2 (PLD2) activity and an induction of receptor endocytosis, whereas the agonist morphine which does not induce opioid receptor endocytosis fails to activate PLD2. We report here that MOPr-mediated activation of PLD2 stimulates production of reactive oxygen molecules via NADH/NADPH oxidase. Oxidative stress was measured with the fluorescent probe dichlorodihydrofluorescein diacetate and the role of PLD2 was assessed by the PLD inhibitor D-erythro-sphingosine (sphinganine) and by PLD2-small interfering RNA transfection. To determine whether NADH/NADPH oxidase contributes to opioid-induced production of reactive oxygen species, mu-agonist-stimulated cells were pre-treated with the flavoprotein inhibitor, diphenylene iodonium, or the specific NADPH oxidase inhibitor, apocynin. Our results demonstrate that receptor-internalizing agonists (like DAMGO, beta-endorphin, methadone, piritramide, fentanyl, sufentanil, and etonitazene) strongly induce NADH/NADPH-mediated ROS synthesis via PLD-dependent signaling pathways, whereas agonists that do not induce MOPr endocytosis and PLD2 activation (like morphine, buprenorphine, hydromorphone, and oxycodone) failed to activate ROS synthesis in transfected human embryonic kidney 293 cells. These findings indicate that the agonist-selective PLD2 activation plays a key role in the regulation of NADH/NADPH-mediated ROS formation by opioids.

Based on a pharmacophore alignment on a 5-HT6 ligand applying 4SCan® technology, a new lead series was identified and further structurally investigated. Kis down to 8 nM were achieved.

Objective: Effects of cannabinoids are mediated by CB1 and CB2 receptors. In addition to neuronal effects, cannabinoids are potent modulators of immune functions. In this report, we investigated whether the transcription of these receptors is regulated after activation of T lymphocytes. Methods: CB1- and CB2-specific mRNA of primary human peripheral blood T cells and cells of the human T cell line Jurkat was measured by quantitative real-time RT-PCR in response to CD3/28. Using the decoy oligonucleotide approach, transcription factors involved in the regulation were determined. A promoter analysis was performed using transient transfection of chloramphenicol acetyl transferase reporter gene constructs in Jurkat cells. Results: Activation of human T cells caused an induction of CB1 mRNA expression in primary human T cells (8-fold) and Jurkat cells (29-fold). In contrast, CB2 transcription was not regulated. The CD3/28-mediated upregulation of CB1 involves the transcription factors AP-1, NFĸB and NFAT. Furthermore, 2,490 bp of the CB1 promoter mediated inducibility in response to CD3/28. Conclusions: The upregulation of CB1 in activated T cells, together with the constitutive expression of CB2, enables cellular responses to cannabinoids mediated by both receptor subtypes. It may thus contribute to the understanding of the various modulatory effects of cannabinoids on activated T cells.

Cannabinoid CB1 receptor, the molecular target of endocannabinoids and cannabis active components, is one of the most abundant metabotropic receptors in the brain. Cannabis is widely used for both recreational and medicinal purposes. Despite the ever-growing fundamental roles of microRNAs in the brain, the possible molecular connections between the CB1 receptor and microRNAs are surprisingly unknown. Here, by using reporter gene constructs that express interaction sequences for microRNAs in human SH-SY5Y neuroblastoma cells, we show that CB1 receptor activation enhances the expression of several microRNAs, including let-7d. This was confirmed by measuring hsa-let-7d expression levels. Accordingly, knocking-down CB1 receptor in zebrafish reduced dre-let-7d levels, and knocking-out CB1 receptor in mice decreased mmu-let-7d levels in the cortex, striatum and hippocampus. Conversely, knocking-down let-7d increased CB1 receptor mRNA expression in zebrafish, SH-SY5Y cells and primary striatal neurons. Likewise, in primary striatal neurons chronically exposed to a cannabinoid or opioid agonist, a let-7d-inhibiting sequence facilitated not only cannabinoid or opioid signaling but also cannabinoid/opioid cross-signaling. Taken together, these findings provide the first evidence for a bidirectional link between the CB1 receptor and a microRNA, namely let-7d, and thus unveil a new player in the complex process of cannabinoid action.

Effects of cannabinoids (CBs) are mediated by two types of receptors, CB1 and CB2. In this report, we investigated whether CBs regulate gene expression of their cognate receptors in T cells and studied underlying mechanisms in CD4+ Jurkat T cells. Transcription of the CB1 gene was strongly induced in response to Δ9-tetrahydroannabinol (THC), whereas the CB2 gene was not regulated. The induction of CB1 gene expression is mediated by CB2 receptors only, as demonstrated by using the CB1 and CB2 agonists R(+)-methanandamide and JWH 015, respectively, and combinations of THC plus CB1- and CB2-specific antagonists. After activation of CB2 receptors, the transcription factor STAT5 is phosphorylated. STAT5 then transactivates IL-4. Induction of IL-4 mRNA as well as IL-4 protein release from the cells are necessary for the following induction of the CB1 gene. This was demonstrated by using decoy oligonucleotides against STAT5, which blocked IL-4 and CB1 mRNA induction, and by using the IL-4 receptor antagonist IL-4 [R121D, Y124D], which blocked the up-regulation of CB1 gene transcription. Transactivation of the CB1 gene in response to IL-4 is then mediated by the transcription factor STAT6, as shown by using decoy oligonucleotides against STAT6. An increase in CB1-mediated phosphorylation of MAPK in cells prestimulated with CB2-specific agonists suggests up-regulation of functional CB1 receptor proteins. In summary, up-regulation of CB1 in T lymphocytes in response to CBs themselves may facilitate or enhance the various immunomodulatory effects related to CBs.

BACKGROUND: Alterations of synapsis can disturb or arrest meiosis and result in infertility. Synaptic abnormalities are frequently observed in infertile patients but also in fertile men. METHODS: The subtelomere-specific multiplex fluorescence in-situ hybridization (stM-FISH) has been applied in combination with immunofluorescence to identify all synaptonemal complexes (SCs) and to analyse those presenting synaptic anomalies in fertile and infertile men. RESULTS: SCs with heterochromatin blocks other than centromere (noncentromeric heterochromatin) presented a higher frequency of gaps (SC discontinuities) and splits (unsynapsed SC regions) at pachytene, the incidences for 9qh, 1qh, 15p and 21p being the highest ones. Inter-individual variability in the incidence of synaptic anomalies in these regions has been observed. In addition, synaptic anomalies in other SC regions are more frequent in infertile cases than in controls. Clear association of the SC15 and SC21 to the XY pair has been seen. CONCLUSION: Noncentromeric heterochromatic regions are the last to synapse. The inter-individual variation observed in the incidence of gaps and splits in these regions may be explained by the heteromorphism of these regions in the general population. The presence of synaptic anomalies in other SC regions may indicate nuclei with a severely affected synapsis. Noncentromeric heterochromatic regions might play a role in the association of autosomal SC15 and SC21 with the XY pair.

Based on a pharmacophore alignment on known non-competitive mGluR5 inhibitors applying 4SCan® technology, a new lead series was identified and further structurally investigated. Ki’s as low as around 100 nM were achieved.

To compare the neuropsychological deficits of primary progressive multiple sclerosis with those of relapsing-remitting and secondary progressive multiple sclerosis.Sixty-five patients with different clinical courses of MS were neuropsychologically tested for language, attention, memory and executive functions. Discriminant analysis was used to predict the type of clinical course either by clinical variables (age, EDSS and duration of illness) or neuropsychological test results.For single neuropsychological tests, group differences were rare between the progressive courses and the relapsing-remitting course of MS or absent between the progressive courses of MS. However, discriminant analysis correctly identified 87.7 percent of the patients' courses in general, and about 90 percent of the patients with chronic progressive MS.Using discriminant analysis, this study found neuropsychological impairment characteristic for relapsing remitting, secondary progressive and primary progressive patients.

The relevance of mu-opioid systems for central excitability and kindling related disturbed learning performance was underlined by investigations using mu-opioid receptor knockout mice. Mice lacking mu-opioid receptors showed an accelerated kindling development induced by the convulsant drug pentylenetetrazol. Blockade of delta-opioid receptors by naltrindole suppressing kindling development in wild-type animals led to a further acceleration of kindled seizure development in the knockout mice. Mice lacking mu-opioid receptors showed such a low learning performance in the shuttle box, that the kindling induced learning deficit as seen in wild-type mice was not detected. The results were discussed on the basis of receptor binding studies with regard to subtypes of glutamatergic receptors, delta-opioid and somatostatin receptors. An increase in glutamate and somatostatin binding could contribute to the enhanced excitability in the-mu-opioid receptor knockout mice.

Opioids and their receptors are key players in a cross-talk between the nervous and immune systems. For example, the endogenous opioid system is activated during inflammation as a physiological feedback mechanism to attenuate inflammatory pain. Herein, we report that in primary human T lymphocytes, Raji B cells, U937 monocytes, primary human polymorphonuclear leukocytes, and mature dendritic cells, the proinflammatory cytokine tumor necrosis factor induced mu-opioid receptor gene transcription. Transcriptional induction of the gene in immune cells was mediated via tumor necrosis factor receptor type 2. Using selective in vivo disruption of possibly involved transcription factors with decoy oligonucleotides, nuclear factor-kappaB was identified as the factor responsible for induction of the gene in immune cells, whereas activator protein-1 was found to be uninvolved. Nuclear factor-kappaB also mediates up-regulation of mu-opioid receptors in neuronal cells stimulated with tumor necrosis factor. Among six putative nuclear factor-kappaB binding sites on the mu-opioid receptor gene promoter, three cis-active elements at nt -2174, -557, and -207 were identified using transfection experiments of reporter gene constructs, electrophoretic mobility shift assays, and in vivo binding studies with decoy oligonucleotides. An allelic variation within the -557 element significantly reduced its trans-activating potency, which may affect regulation of the mu-opioid receptor gene in persons carrying this mutation. This study suggests a regulatory function of tumor necrosis factor in opioid-mediated processes in neuronal and immune cells, with possible impact on the complex of inflammation-induced analgesia.

A preparative synthesis of novel bioxepines and bi[10]paracyclophanes with meso - and rac -configuration is described. The bi[10]paracyclophane (−)- 6b with two elements of planar chirality and one chiral axis has been obtained in enantiomerically pure form. Its absolute configuration was determined by quantum chemical calculation of the circular dichroism and comparison with the experimental CD spectrum.

Metastasis is responsible for most deaths from cancer. Currently, little is known about the early genetic events in the metastatic evolution. Here we describe the application of a newly developed strategy for an in-depth characterization of genomic changes in micrometastatic cells. Unique tumor cell lines were established from bone marrow of patients with cancer of the prostate and analyzed by multiplex-FISH (M-FISH) and array CGH. M-FISH revealed that the occult disseminated cells were characterized by very complex numerical and structural aberrations. Many of these aberrations resulted in chromosomal gains and losses, such as losses of 8p, 13q, and 18q and gains of 8q, 9q, 20, and the X chromosome, which are typically observed in prostate cancer. Array CGH allowed an unprecedented high-resolution assessment of copy number changes, pinpointing commonly gained or lost regions, which should narrow down the identification of regions critically involved in metastasis. Thus, occult micrometastatic cells are now amenable to detailed analyses of their genome. Markers for prognosis and treatment decisions can now be established.

Supratentorial primitive neuroectodermal tumours (sPNETs) are malignant central nervous system tumours of childhood which are histologically characterized by poorly differentiated neuroepithelial cells with the capacity for divergent differentiation into glial, neuronal, myogenic or melanotic lines. The histological differential diagnosis between sPNET and glioblastoma multiforme (GBM) may be difficult, particularly as GBMs can sometimes demonstrate a poorly differentiated PNET-like phenotype. To identify molecular genetic markers that may distinguish sPNET and GBM, we investigated 12 cerebral sPNETs and six GBMs from paediatric patients for genetic alterations of the TP53, PTEN, CDKN2A, EGFR, CDK4 and MDM2 genes, as well as for allelic loss on chromosome arms 10q and 17p. Mutations of the TP53 tumour suppressor gene were found in one of 12 sPNETs (8%) and two of six GBMs (33%). None of the sPNETs but two of six GBMs (33%, including one GBM with a TP53 mutation) showed allelic losses on chromosome arm 17p. PTEN mutations were detected in one of 12 sPNET (8%) and one of six GBMs (17%). None of the sPNETs and GBMs carried a homozygous deletion involving the CDKN2A tumour suppressor gene. No amplification of the EGFR, CDK4 or MDM2 proto-oncogenes was detected. Taken together, our results indicate that paediatric GBMs differ from sPNETs by a higher incidence of allelic losses on 17p and TP53 mutations. In addition, the patterns of genetic alterations in sPNETs and paediatric GBMs appear to be distinct from those in cerebellar medulloblastomas and adult GBMs, respectively.

The "lactone concept" has been efficiently employed for the first atropo-enantioselective synthesis of knipholone and related natural phenylanthraquinones. Besides the regio- and stereoselective construction of the biaryl axis, another important step was the "synthetically late" introduction of the C-acetyl group, either by a Friedel-Crafts type acetylation or by an ortho-selective Fries rearrangement first tested on simplified model systems and subsequently applied to the highly atroposelective preparation of the natural products and of simplified analogs thereof for biotesting. The synthetic availability of these natural biaryls, their precursors, and their unnatural analogs permitted a broader investigation of the antiplasmodial activities of these interesting biaryls.

Malignant astrocytomas are among the most common brain tumours and few therapeutic options exist. It has recently been recognized that the ligand-activated nuclear receptor PPARgamma can regulate cellular proliferation and induce apoptosis in different malignant cells. We report the effect of three structurally different PPARgamma agonists inducing apoptosis in human (U87MG and A172) and rat (C6) glioma cells. The PPARgamma agonists ciglitazone, LY171 833 and prostaglandin-J2, but not the PPARalpha agonist WY14643, inhibited proliferation and induced cell death. PPARgamma agonist-induced cell death was characterized by DNA fragmentation and nuclear condensation, as well as inhibited by the synthetic receptor-antagonist bisphenol A diglycidyl ether (BADGE). In contrast, primary murine astrocytes were not affected by PPARgamma agonist treatment. The apoptotic death in the glioma cell lines treated with PPARgamma agonists was correlated with the transient up-regulation of Bax and Bad protein levels. Furthermore, inhibition of Bax expression by specific antisense oligonucleotides protected glioma cells against PPARgamma-mediated apoptosis, indicating an essential role of Bax in PPARgamma-induced apoptosis. However, PPARgamma agonists not only induced apoptosis but also caused redifferentiation as indicated by outgrowth of long processes and expression of the redifferentiation marker N-cadherin in response to PPARgamma agonists. Taken together, treatment of glioma cells with PPARgamma agonists may hold therapeutic potential for the treatment of gliomas.

Increased prevalence of small-sized low-density lipoprotein (LDL) subclass B (diameter25.5 nm) possibly is involved in the multifactorial process of cardiovascular disease in patients with end-stage renal disease. Given these epidemiological observations, mechanisms underlying the combined effect of a proinflammatory insult and LDL of different subclasses (subclass A, diameter25.5 nm, and subclass B) in a cellular model were investigated. For this, human umbilical vein endothelial cells were preexposed to LDL, then stimulated with tumor necrosis factor-alpha (TNF-alpha). Modulatory effects of LDL phenotypes on the activation of adhesion molecules, monocyte adherence, and transcriptional activity of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) were investigated. Our data show that subclass B LDLs were metabolized through nonspecific scavenger receptors and specific LDL-receptor pathways in endothelial cells. Furthermore, LDL subclass B in comparison to subclass A more effectively enhanced monocyte recruitment and adhesive properties of endothelial cells in response to TNF-alpha. These effects appeared not to be mediated by oxidative stress-responsive NF-kappaB because modulation of this transcription factor by LDL was moderate and similar for both LDL phenotypes. Conversely, effects of LDL subclass B were considered to be caused by augmented AP-1 binding activity. In conclusion, the present model provides new clues in atherogenic mechanisms of small-sized LDLs, which sensitize vascular cells to inflammatory signals more effectively than normal-sized LDLs.

The atropo-enantioselective total synthesis of the axially chiral bicoumarin (+)-isokotanin A (1) is described. Key steps were the formation of a configurationally stable seven-membered biaryl lactone and its kinetic resolution by atroposelective ring cleavage. The previous assignment of the absolute configuration of (+)-isokotanin A (1) (and its synthetic precursors) was confirmed by quantum chemical CD calculations. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)