SCFFbxw5 targets kinesin-13 proteins to facilitate ciliogenesis.

Microtubule depolymerases of the kinesin-13 family play important roles in various cellular processes and are frequently overexpressed in different cancer types. Despite the importance of their correct abundance, remarkably little is known about how their levels are regulated in cells. Using comprehensive screening on protein microarrays, we identified 161 candidate substrates of the multi-subunit ubiquitin E3 ligase SCFFbxw5 , including the kinesin-13 member Kif2c/MCAK. In vitro reconstitution assays demonstrate that MCAK and its closely related orthologs Kif2a and Kif2b become efficiently polyubiquitylated by neddylated SCFFbxw5 and Cdc34, without requiring preceding modifications. In cells, SCFFbxw5  targets MCAK for proteasomal degradation predominantly during G2 . While this seems largely dispensable for mitotic progression, loss of Fbxw5 leads to increased MCAK levels at basal bodies and impairs ciliogenesis in the following G1 /G0 , which can be rescued by concomitant knockdown of MCAK, Kif2a or Kif2b. We thus propose a novel regulatory event of ciliogenesis that begins already within the G2 phase of the preceding cell cycle.

Valorization of Unconventional Lipids from Microalgae or Tall Oil via a Selective Dual Catalysis One-Pot Approach.

A dual catalysis approach enables selective functionalization of unconventional feedstocks composed of complex fatty acid mixtures with highly unsaturated portions like eicosapentaenoate (20:5) along with monounsaturated compounds. The degree of unsaturation is unified by selective heterogeneous hydrogenation on Pd/γ-Al2O3, complemented by effective activation to a homogeneous carbonylation catalyst [(dtbpx)PdH(L)]+ by addition of diprotonated diphosphine (dtbpxH2)(OTf)2. By this one-pot approach, neat 20:5 as a model substrate is hydrogenated to up to 80% to the monounsaturated analogue (20:1), this is functionalized to the desired C21 α,ω-diester building block with a linear selectivity of over 90%. This catalytic approach is demonstrated to be suitable for crude microalgae oil from Phaeodactylum tricornutum genetically engineered for this purpose, as well as tall oil, an abundant waste material. Both substrates were fully converted with an overall selectivity to the linear α,ω-diester of up to 75%.

Synthetic polyester from algae oil.

Current efforts to technically use microalgae focus on the generation of fuels with a molecular structure identical to crude oil based products. Here we suggest a different approach for the utilization of algae by translating the unique molecular structures of algae oil fatty acids into higher value chemical intermediates and materials. A crude extract from a microalga, the diatom Phaeodactylum tricornutum, was obtained as a multicomponent mixture containing amongst others unsaturated fatty acid (16:1, 18:1, and 20:5) phosphocholine triglycerides. Exposure of this crude algae oil to CO and methanol with the known catalyst precursor [{1,2-(tBu2 PCH2)2C6H4}Pd(OTf)](OTf) resulted in isomerization/methoxycarbonylation of the unsaturated fatty acids into a mixture of linear 1,17- and 1,19-diesters in high purity (>99 %). Polycondensation with a mixture of the corresponding diols yielded a novel mixed polyester-17/19.17/19 with an advantageously high melting and crystallization temperature.

ADAM15 exerts an antiapoptotic effect on osteoarthritic chondrocytes via up-regulation of the X-linked inhibitor of apoptosis.

OBJECTIVE: To investigate the capacity of ADAM15, a disintegrin metalloproteinase that is up-regulated in osteoarthritic (OA) cartilage, to protect chondrocytes against apoptosis induced by growth factor deprivation and genotoxic stress. METHODS: Caspase 3/7 activity was determined in primary OA and ADAM15-transfected T/C28a4 chondrocytes upon exposure to the DNA-damaging agent camptothecin or serum withdrawal. Camptothecin-induced cytotoxicity was determined by measuring cellular ATP content. (Anti-)apoptotic proteins were analyzed by immunoblotting, and levels of messenger RNA (mRNA) for X-linked inhibitor of apoptosis (XIAP) were determined using real-time polymerase chain reaction. RNA interference was applied for down-regulation of ADAM15 and XIAP expression. Immunohistochemistry analysis of normal and OA cartilage samples was performed using XIAP- and ADAM15-specific antibodies. RESULTS: ADAM15-transfected chondrocytes cultured on a collagen matrix displayed significantly reduced caspase 3/7 activity upon serum or intermittent matrix withdrawal, compared with vector-transfected control cells. Apoptosis induction by camptothecin exposure also led to significantly elevated caspase 3/7 activity and reduced cell viability of the vector-transfected compared with ADAM15-transfected chondrocytes. Increased levels of activated caspase 3 and cleaved poly(ADP-ribose) polymerase were detected in the vector controls. XIAP, an inhibitor of activated caspase 3, was significantly up-regulated ( approximately 3-fold) at the protein and mRNA levels in ADAM15-transfected chondrocytes upon camptothecin treatment. Specific down-regulation of either ADAM15 or XIAP in OA chondrocytes led to significant sensitization to camptothecin-induced caspase 3/7 activity. Immunohistochemical analysis revealed low to moderate XIAP expression in normal specimens and markedly increased XIAP staining, colocalizing with ADAM15, in OA cartilage. CONCLUSION: ADAM15 conveys antiapoptotic properties to OA chondrocytes that might sustain their potential to better resist the influence of death-inducing stimuli under pathophysiologic conditions.

Quantitative detection of human adenovirus from river water by monolithic adsorption filtration and quantitative PCR.

Water contaminated with fecally derived viruses, also known as enteric viruses, represents a particularly high risk for human health. However, they have not been included in water quality regulations yet. The detection of these viruses is often more expensive and time-consuming compared to the analysis of conventional fecal indicator organisms. In addition, most methods are not sensitive enough to detect small viral loads that may already cause serious health issues if present in water. In this study, we established a workflow for the successful and direct enrichment of human adenovirus (HAdV) from artificially contaminated river water based on monolithic adsorption filtration (MAF) and quantitative polymerase reaction (qPCR). With a clear focus on efficiency, we used targeted synthetic DNA fragments as standard for the quantification of HAdV by qPCR, leading to accurate and robust results with a qPCR efficiency of 95 %, a broad working range over 6 orders of magnitude and an LOD of 1 GU/µL. We carried out a cascade of spiking experiments, enhancing the complexity of the spiking matrix with each step to progressively evaluate MAF for the direct concentration of HAdV. We found that negatively charged MAF using monoliths with hydroxyl groups (MAF-OH) showed a better reproducibility and a significantly faster turnaround time than skimmed milk flocculation (SMF) when concentrating HAdV35 from artificially contaminated, acidified mineral water. We then validated positively charged MAF using monoliths with diethyl aminoethyl groups (MAF-DEAE) for the direct concentration of HAdV5 without pre-conditioning of water samples using tap water as spiking matrix with a less defined and controlled water chemistry. Finally, we evaluated MAF-DEAE for the direct concentration of HAdV5 from surface water using river water as representative matrix with an undefined water chemistry. We found, that MAF-DEAE achieved reproducible recoveries of HAdV5, independently of the spiked concentration level or sample volume. Furthermore, we showed, that MAF-DEAE drastically reduced the limit of detection (LOD) of HAdV5 by a factor of 115 from 6.0 ∙ 103 GU/mL before to 5.2 ∙ 101 GU/mL after MAF-DEAE. We identified that recoveries increased for smaller processing volumes with a peak at 0.5 L of 84.0 % and showed that recovery efficiency depends on sample volume and matrix type. The here presented workflow based on MAF-DEAE and qPCR offers an easy-to-implement and highly efficient alternative to existing approaches and allows for a fast detection of HAdV in water.

Interferon-stimulated gene 15 accelerates replication fork progression inducing chromosomal breakage.

DNA replication is highly regulated by the ubiquitin system, which plays key roles upon stress. The ubiquitin-like modifier ISG15 (interferon-stimulated gene 15) is induced by interferons, bacterial and viral infection, and DNA damage, but it is also constitutively expressed in many types of cancer, although its role in tumorigenesis is still largely elusive. Here, we show that ISG15 localizes at the replication forks, in complex with PCNA and the nascent DNA, where it regulates DNA synthesis. Indeed, high levels of ISG15, intrinsic or induced by interferon-ß, accelerate DNA replication fork progression, resulting in extensive DNA damage and chromosomal aberrations. This effect is largely independent of ISG15 conjugation and relies on ISG15 functional interaction with the DNA helicase RECQ1, which promotes restart of stalled replication forks. Additionally, elevated ISG15 levels sensitize cells to cancer chemotherapeutic treatments. We propose that ISG15 up-regulation exposes cells to replication stress, impacting genome stability and response to genotoxic drugs.

Opposite effects of anandamide and N-arachidonoyl dopamine in the regulation of prostaglandin E and 8-iso-PGF formation in primary glial cells.

It is widely accepted that neuroinflammation is a key player in various pathological events associated with brain injury. More specifically, glial activation and the subsequent release of pro-inflammatory cytokines, reactive oxygen species (ROS), and prostaglandins play a role of paramount importance in cerebral damage. In this study, we examined the role of two endocannabinoids, anandamide (AEA) and N-arachidonoyldopamine (NADA) in the regulation of prostaglandin E(2) (PGE(2)) synthesis in primary glial cells. We show that NADA is a potent inhibitor of PGE(2) synthesis in lipopolysaccharide (LPS) stimulated cells, without modifying the expression or enzymatic activity of COX-2 and the production of prostaglandin D(2). We also show that NADA has the ability to prevent the free radical formation in primary microglial cells. The key findings of this investigation are our observation that AEA and NADA have opposite effects on glial cells and, most importantly, the first description of NADA as a potential antioxidative and anti-inflammatory agent acting through a mechanism that involves reduction in the synthesis of microsomal prostaglandin E synthase in LPS-activated microglia. These findings provide new mechanistic insights into the anti-inflammatory activities of NADA in the CNS and its potential to design novel therapeutic strategies to manage neuroinflammatory diseases.

Detection of Meningeosis neoplastica by real-time quantitation of telomerase activity.

BACKGROUND: Analysis of cerebrospinal fluid (CSF) to discriminate between benign and malignant conditions is of fundamental importance for the physician and the patient because of the differential therapeutic options and resulting morbidity and mortality. Most human tumours demonstrate increased telomerase activity (TA). Recent technical advances in the detection of TA allow for sensitive and specific detection within 4 h. Thus, the detection of TA is suitable for routine clinical testing. METHODS: This study examines TA in cellular proteins in CSF from 111 patients compared to cytomorphological and laboratory examination. RESULTS: A positive result for TA in cellular proteins of CSF was correlated significantly with Meningeosis neoplastica, but not with non-malignant conditions. Telomerase was not detected in CSF supernatant, despite positive results in cellular proteins from identical patients. Furthermore, a 48-h time delay during the pre-analytic processing is not critical for detection of TA detection in native CSF when stored at room temperature. CONCLUSIONS: We conclude that TA is a promising marker for the detection of Meningeosis neoplastica and warrants further study.

Strategies to Target ISG15 and USP18 Toward Therapeutic Applications.

The interferon (IFN)-stimulated gene product 15 (ISG15) represents an ubiquitin-like protein (Ubl), which in a process termed ISGylation can be covalently linked to target substrates via a cascade of E1, E2, and E3 enzymes. Furthermore, ISG15 exerts functions in its free form both, as an intracellular and as a secreted protein. In agreement with its role as a type I IFN effector, most functions of ISG15 and ISGylation are linked to the anti-pathogenic response. However, also key roles in other cellular processes such as protein translation, cytoskeleton dynamics, exosome secretion, autophagy or genome stability and cancer were described. Ubiquitin-specific protease 18 (USP18) constitutes the major ISG15 specific protease which counteracts ISG15 conjugation. Remarkably, USP18 also functions as a critical negative regulator of the IFN response irrespective of its enzymatic activity. Concordantly, lack of USP18 function causes fatal interferonopathies in humans and mice. The negative regulatory function of USP18 in IFN signaling is regulated by various protein-protein interactions and its stability is controlled via proteasomal degradation. The broad repertoire of physiological functions and regulation of ISG15 and USP18 offers a variety of potential intervention strategies which might be of therapeutic use. Due to the high mutation rates of pathogens which are often species specific and constantly give rise to a variety of immune evasion mechanisms, immune effector systems are under constant evolutionarily pressure. Therefore, it is not surprising that considerable differences in ISG15 with respect to function and sequence exist even among closely related species. Hence, it is essential to thoroughly evaluate the translational potential of results obtained in model organisms especially for therapeutic strategies. This review covers existing and conceptual assay systems to target and identify modulators of ISG15, ISGylation, USP18 function, and protein-protein interactions within this context. Strategies comprise mouse models for translational perspectives, cell-based and biochemical assays as well as chemical probes.

The acetaminophen-derived bioactive N-acylphenolamine AM404 inhibits NFAT by targeting nuclear regulatory events.

AM404 is a synthetic TRPV1/CB(1) hybrid ligand with inhibitory activity on the anandamide transporter and is used for the pharmacological manipulation of the endocannabinoid system. It has been recently described that acetaminophen is metabolised in the brain to form the bioactive N-acylphenolamine AM404 and therefore, we have evaluated the effect of this metabolite in human T cells, discovering that AM404 is a potent inhibitor of TCR-mediated T-cell activation. Moreover, we found that AM404 specifically inhibited both IL-2 and TNF-alpha gene transcription and TNF-alpha synthesis in CD3/CD28-stimulated Jurkat T cells in a FAAH independent way. To further characterize the biochemical inhibitory mechanisms of AM404, we examined the signaling pathways that regulate the activation of the transcription factors NF-kappaB, NFAT and AP-1 in Jurkat cells. We found that AM404 inhibited both the binding to DNA and the transcriptional activity of endogenous NFAT and the transcriptional activity driven by the over expressed fusion protein Gal4-NFAT (1-415). However, AM404 did not affect early steps in NFAT signaling such as CD3-induced calcium mobilization and NFAT1 dephosphorylation. The NFAT inhibitory activity of AM404 seems to be quite specific since this compound did not interfere with the signaling pathways leading to AP-1 or NF-kappaB activation. These findings provide new mechanistic insights into the immunological effects of AM404 which in part could explain some of the activities ascribed to the widely used acetaminophen.

Structural basis of the specificity of USP18 toward ISG15.

Protein modification by ubiquitin and ubiquitin-like modifiers (Ubls) is counteracted by ubiquitin proteases and Ubl proteases, collectively termed DUBs. In contrast to other proteases of the ubiquitin-specific protease (USP) family, USP18 shows no reactivity toward ubiquitin but specifically deconjugates the interferon-induced Ubl ISG15. To identify the molecular determinants of this specificity, we solved the crystal structures of mouse USP18 alone and in complex with mouse ISG15. USP18 was crystallized in an open and a closed conformation, thus revealing high flexibility of the enzyme. Structural data, biochemical and mutational analysis showed that only the C-terminal ubiquitin-like domain of ISG15 is recognized and essential for USP18 activity. A critical hydrophobic patch in USP18 interacts with a hydrophobic region unique to ISG15, thus providing evidence that USP18's ISG15 specificity is mediated by a small interaction interface. Our results may provide a structural basis for the development of new drugs modulating ISG15 linkage.

High-resolution gadolinium-enhanced 3D MRA of the infrapopliteal arteries. Lessons for improving bolus-chase peripheral MRA.

Peripheral magnetic resonance angiography (MRA) is growing in use. However, methods of performing peripheral MRA vary widely and continue to be optimized, especially for improvement in illustration of infrapopliteal arteries. The main purpose of this project was to identify imaging factors that can improve arterial visualization in the lower leg using bolus chase peripheral MRA. Eighteen healthy adults were imaged on a 1.5T MR scanner. The calf was imaged using conventional three-station bolus chase three-dimensional (3D) MRA, two dimensional (2D) time-of-flight (TOF) MRA and single-station Gadolinium (Gd)-enhanced 3D MRA. Observer comparisons of vessel visualization, signal to noise ratios (SNR), contrast to noise ratios (CNR) and spatial resolution comparisons were performed. Arterial SNR and CNR were similar for all three techniques. However, arterial visualization was dramatically improved on dedicated, arterial-phase Gd-enhanced 3D MRA compared with the multi-station bolus chase MRA and 2D TOF MRA. This improvement was related to optimization of Gd-enhanced 3D MRA parameters (fast injection rate of 2 mL/sec, high spatial resolution imaging, the use of dedicated phased array coils, elliptical centric k-space sampling and accurate arterial phase timing for image acquisition). The visualization of the infrapopliteal arteries can be substantially improved in bolus chase peripheral MRA if voxel size, contrast delivery, and central k-space data acquisition for arterial enhancement are optimized. Improvements in peripheral MRA should be directed at these parameters.

Investigation of Oxygen Permeation through Composites of PMMA and Surface-Modified ZnO Nanoparticles.

Oxygen permeabilities of nanocomposite films consisting of poly(methyl methacrylate) (PMMA) and different amounts of spherical zinc oxide (ZnO) nanoparticles were determined to investigate the barrier effect of this material with respect to particle content. A method was applied which is based on quenching of an excited phosphorescent dye by oxygen. Possible effects of the nanoparticles on the response of the dye molecules were investigated and were ruled out.

Self-assembled structures in organogels of amphiphilic diblock codendrimers.

Amphiphilic diblock codendrimers consisting of dendrons of hydroxyl-containing poly(methallyl dichloride) (PMDC) and long alkyl-containing poly(urethane amide) (PUA) were synthesized in different generations. These codendrimers were found to self-assemble into ribbonlike aggregates in organic solvent and further formed three-dimensional networks and behaved macroscopically as gels. The width of the self-assembled ribbons decreases with the generation of both dendritic blocks. Multiple intermolecular hydrogen bonds between amide and hydroxyl groups were found to be the main driving force to form these self-assembled gels.

Signal transduction pathways regulating cyclooxygenase-2 in lipopolysaccharide-activated primary rat microglia.

Microglia are the major cell type involved in neuroinflammatory events in brain diseases such as encephalitis, stroke, and neurodegenerative disorders, and contribute significantly to the release of prostaglandins (PGs) during neuronal insults. In this report, we studied the immediate-early intracellular signalling pathways in microglia, following bacterial lipopolysaccharide (LPS) stimulation, leading to the synthesis and release of PGE2. Here we show that LPS induces cyclooxygenase (COX) 2 by activating sphingomyelinases leading to the release of ceramides, which in turn, activate the p38 mitogen-activated protein kinases (MAPK), but not the p42/44 MAPK. We further show that exogenously added ceramide analogue (C2-ceramide) also induce PGE2 synthesis through a p38 MAPK-dependent pathway. This potential nature of ceramides in activating microglia suggests that endogenously produced ceramides during neuronal apoptosis in ischemia or neurodegenerative diseases could also contribute to the amplification of neuroinflammatory events. In contrast to protein kinase C (PKC) and phosphocholine-specific phospholipase C (PC-PLC), which transcriptionally regulate LPS-induced COX-2 synthesis, inhibition of phospholipase A2 (PLA2) has no effect on COX-2 transcription, although it inhibits the release of PGE2. Transcriptional regulation of LPS-induced COX-2 by PKC is further proved by the ability of the PKC inhibitor, Gö 6976, to inhibit LPS-induced 8-isoprostane synthesis, but not affecting LPS-induced COX-2 activity. Our data with 8-isoprostane also indicates that COX-2 plays a major role in ROS production in LPS-activated microglia. This detailed view of the intracellular signaling pathway in microglial activation and COX-2 expression opens a new therapeutic window in the search for new and more effective central anti-inflammatory agents.

Polymeric membranes for aromatic/aliphatic separation processes.

For the membrane-based separation of benzene/cyclohexane mixtures, the pervaporation properties of different 6FDA (4,4'-hexafluoroisopropylidene diphthalic anhydride) based copolyimide membranes have been investigated. In order to obtain high permeability as well as high selectivity copolyimides were synthesised using a combination of 4MPD (2,3,5,6-tetramethyl-1,4-phenylene diamine) and 6FpDA (4,4'-hexafluoro-isopropylidene dianiline) as well as DABA (3,5-diaminobenzoic acid) as monomers. Cross-linking is possible with this type of copolyimides and necessary to reduce swelling effects, which often occur with polymeric membranes and lead to a deterioration of the separation characteristics in aromatic/aliphatic separation processes. In order to find the most suitable membrane material, the polymer structure, the crosslinking agents, as well as the crosslinking methods have been varied. The separation characteristics have been determined by sorption and pervaporation experiments. Sorption experiments have been carried out at 60 degrees C using benzene, toluene and ethylbenzene as aromatic components and cyclohexane, cyclohexene, hexane and heptane as aliphatic solvents. Pervaporation experiments have been performed at 60 degrees C using benzene/cyclohexane mixtures with benzene concentrations covering the whole concentration range. It has been found that crosslinked copolyimide membranes show excellent chemical resistance, strongly reduced swelling behaviour and higher selectivity in aromatic/aliphatic separation compared to conventional membrane materials.