Identification of oxidation sites and covalent cross-links in metal catalyzed oxidized interferon Beta-1a: potential implications for protein aggregation and immunogenicity.

Oxidation via Cu(2+)/ascorbate of recombinant human interferon beta-1a (IFNβ1a) leads to highly immunogenic aggregates, however it is unknown which amino acids are modified and how covalent aggregates are formed. In the present work we mapped oxidized and cross-linked amino acid residues in aggregated IFNβ1a, formed via Cu(2+)/ascorbate catalyzed oxidation. Size exclusion chromatography (SEC) was used to confirm extensive aggregation of oxidized IFNβ1a.

Two populations of glucocorticoid receptor-binding sites in the male rat hippocampal genome.

In the present study, genomic binding sites of glucocorticoid receptors (GR) were identified in vivo in the rat hippocampus applying chromatin immunoprecipitation followed by next-generation sequencing. We identified 2470 significant GR-binding sites (GBS) and were able to confirm GR binding to a random selection of these GBS covering a wide range of P values. Analysis of the genomic distribution of the significant GBS revealed a high prevalence of intragenic GBS.

High density lipoprotein as a source of cholesterol for adrenal steroidogenesis: a study in individuals with low plasma HDL-C.

Few studies have addressed the delivery of lipoprotein-derived cholesterol to the adrenals for steroid production in humans. While there is evidence against a role for low-density lipoprotein (LDL), it is unresolved whether high density lipoprotein (HDL) contributes to adrenal steroidogenesis. To study this, steroid hormone profiles in urine were assessed in male subjects suffering from functional mutations in ATP binding cassette transporter A1 (ABCA1) (n = 24), lecithin:cholesterol acyltransferase (LCAT) (n = 40), as well as in 11 subjects with low HDL cholesterol (HDL-C) without ABCA1/LCAT mutations.

Characterization and skin permeation of ketoprofen-loaded vesicular systems.

Purpose: To determine the effect of elasticity on the skin permeation of ketoprofen from surfactant-based vesicular formulations and elastic liposomes.

Methods: Ketoprofen-loaded surfactant-based vesicles and elastic liposomes were prepared by sonication. Citric buffer (at pH 3.

Significantly improved HIV inhibitor efficacy prediction employing proteochemometric models generated from antivirogram data.

Infection with HIV cannot currently be cured; however it can be controlled by combination treatment with multiple anti-retroviral drugs. Given different viral genotypes for virtually each individual patient, the question now arises which drug combination to use to achieve effective treatment. With the availability of viral genotypic data and clinical phenotypic data, it has become possible to create computational models able to predict an optimal treatment regimen for an individual patient.

Utility of CSF in translational neuroscience.

Human cerebrospinal fluid (CSF) sampling is of high value as the only general applicable methodology to obtain information on free drug concentrations in individual human brain. As the ultimate interest is in the free drug concentration at the CNS target site, the question is what CSF concentrations may tell us in that respect. Studies have been performed in rats and other animals for which concentrations in brain extracellular fluid (brain ECF) as a target site for many drugs, have been compared to (cisterna magna) CSF concentrations, at presumed steady state conditions,.

From heptahelical bundle to hits from the Haystack: structure-based virtual screening for GPCR ligands.

This review will focus on the construction, refinement, and validation of G-protein-coupled receptor (GPCR) structural models for the purpose of structure-based virtual screening (SBVS) and ligand design. The review will present a comparative analysis of GPCR crystal structures and their implication on GPCR (homology) modeling. The challenges associated with steps along the modeling workflow will be discussed: the use of experimental anchors to steer the modeling procedure, amino acid sequence alignment and template selection, receptor structure refinement, loop modeling, ligand-binding mode prediction, and virtual screening for novel ligands.

Systems biology approach identifies the kinase Csnk1a1 as a regulator of the DNA damage response in embryonic stem cells.

In pluripotent stem cells, DNA damage triggers loss of pluripotency and apoptosis as a safeguard to exclude damaged DNA from the lineage. An intricate DNA damage response (DDR) signaling network ensures that the response is proportional to the severity of the damage. We combined an RNA interference screen targeting all kinases, phosphatases, and transcription factors with global transcriptomics and phosphoproteomics to map the DDR in mouse embryonic stem cells treated with the DNA cross-linker cisplatin.

Identifying the translational gap in the evaluation of drug-induced QTc interval prolongation.

Aims: Given the similarities in QTc response between dogs and humans, dogs are used in pre-clinical cardiovascular safety studies. The objective of our investigation was to characterize the PKPD relationships and identify translational gaps across species following the administration of three compounds known to cause QTc interval prolongation, namely cisapride, d, l-sotalol and moxifloxacin.

Methods: Pharmacokinetic and pharmacodynamic data from experiments in conscious dogs and clinical trials were included in this analysis.

Lack of compliance of European Public Assessment Reports to guidelines for paediatric drug development before the introduction of paediatric investigation plans.

Background: According to the International Conference on Harmonisation (ICH) and Food and Drug Administration (FDA) guidelines for paediatric clinical trials, bridging procedures can be used if disease progression, exposure-response relationships, and clinical endpoints are similar in adults and children. In these circumstances, confirmatory efficacy trials are not necessary; the evaluation of pharmacokinetics and safety ought to be sufficient for drug approval.

Purpose: The aim of this study was to assess whether the clinical trials and strategy for market approval authorisation (MAAs) in paediatric indications reflect the guidelines for bridging of adult data.

Automated analysis of NF-κB nuclear translocation kinetics in high-throughput screening.

Nuclear entry and exit of the NF-κB family of dimeric transcription factors plays an essential role in regulating cellular responses to inflammatory stress. The dynamics of this nuclear translocation can vary significantly within a cell population and may dramatically change e.g.

Characterization of human cytochrome P450s involved in the bioactivation of clozapine.

Clozapine is known to cause hepatotoxicity in a small percentage of patients. Oxidative bioactivation to reactive intermediates by hepatic cytochrome P450s (P450s) has be proposed as a possible mechanism. However, in contrast to their role in formation of N-desmethylclozapine and clozapine N-oxide, the involvement of individual P450s in the bioactivation to reactive intermediates is much less well characterized.

Antisense-mediated isoform switching of steroid receptor coactivator-1 in the central nucleus of the amygdala of the mouse brain.

Background: Antisense oligonucleotide (AON)-mediated exon skipping is a powerful tool to manipulate gene expression. In the present study we investigated the potential of exon skipping by local injection in the central nucleus of the amygdala (CeA) of the mouse brain. As proof of principle we targeted the splicing of steroid receptor coactivator-1 (SRC-1), a protein involved in nuclear receptor function.

Stress and estrous cycle affect strategy but not performance of female C57BL/6J mice.

Stress induces a switch in learning strategies of male C57BL/6J mice from predominantly spatial to more stimulus-response learning. To study generalization of these findings over sex, we investigated female C57BL/6J mice at three phases of the estrous cycle under non stress and acute (10 min) restraint stress conditions. On a circular hole board (CHB) task, about half of the naive female mice used spatial and stimulus-response strategies to solve the task.

Adrenal-specific scavenger receptor BI deficiency induces glucocorticoid insufficiency and lowers plasma very-low-density and low-density lipoprotein levels in mice.

Objective: We determined the physiological consequences of adrenocortical-specific deletion of scavenger receptor BI (SR-BI) function in C57BL/6 wild-type mice.

Methods And Results: One adrenal from 10-day-old SR-BI knockout (KO) mice or wild-type controls was transplanted under the renal capsule of adrenalectomized C57BL/6 recipient mice. The fasting plasma corticosterone level increased over time in transplanted mice.

Optimization of encapsulation of a synthetic long peptide in PLGA nanoparticles: low-burst release is crucial for efficient CD8(+) T cell activation.

Overlapping synthetic long peptides (SLPs) hold great promise for immunotherapy of cancer. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) are being developed as delivery systems to improve the potency of peptide-based therapeutic cancer vaccines. Our aim was to optimize PLGA NP for SLP delivery with respect to encapsulation and release, using OVA24, a 24-residue long synthetic antigenic peptide covering a CTL epitope of ovalbumin (SIINFEKL), as a model antigen.

Assessing the metabolic effects of prednisolone in healthy volunteers using urine metabolic profiling.

Background: Glucocorticoids, such as prednisolone, are widely used anti-inflammatory drugs, but therapy is hampered by a broad range of metabolic side effects including skeletal muscle wasting and insulin resistance. Therefore, development of improved synthetic glucocorticoids that display similar efficacy as prednisolone but reduced side effects is an active research area. For efficient development of such new drugs, in vivo biomarkers, which can predict glucocorticoid metabolic side effects in an early stage, are needed.

LCAT deficiency in mice is associated with a diminished adrenal glucocorticoid function.

In vitro studies have suggested that HDL and apoB-containing lipoproteins can provide cholesterol for synthesis of glucocorticoids. Here we assessed adrenal glucocorticoid function in LCAT knockout (KO) mice to determine the specific contribution of HDL-cholesteryl esters to adrenal glucocorticoid output in vivo. LCAT KO mice exhibit an 8-fold higher plasma free cholesterol-to-cholesteryl ester ratio (P < 0.

Identification and profiling of CXCR3-CXCR4 chemokine receptor heteromer complexes.

Background And Purpose: The C-X-C chemokine receptors 3 (CXCR3) and C-X-C chemokine receptors 4 (CXCR4) are involved in various autoimmune diseases and cancers. Small antagonists have previously been shown to cross-inhibit chemokine binding to CXCR4, CC chemokine receptors 2 (CCR2) and 5 (CCR5) heteromers. We investigated whether CXCR3 and CXCR4 can form heteromeric complexes and the binding characteristics of chemokines and small ligand compounds to these chemokine receptor heteromers.

Nature versus nurture: does human skin maintain its stratum corneum lipid properties in vitro?

Human skin equivalents (HSEs) mimic human skin closely, but show differences in their stratum corneum (SC) lipid properties. The aim of this study was to determine whether isolation of primary cells, which is needed to generate HSEs, influence the SC lipid properties of HSEs. For this purpose, we expanded explants of intact full thickness human skin and isolated epidermal sheets in vitro.

Chemical subtleties in small-molecule modulation of peptide receptor function: the case of CXCR3 biaryl-type ligands.

The G protein-coupled chemokine receptor CXCR3 plays a role in numerous inflammatory events. The endogenous ligands for the chemokine receptors are peptides, but in this study we disclose small-molecule ligands that are able to activate CXCR3. A class of biaryl-type compounds that is assembled by convenient synthetic routes is described as a new class of CXCR3 agonists.

Constitutive β-catenin signaling by the viral chemokine receptor US28.

Chronic activation of Wnt/β-catenin signaling is found in a variety of human malignancies including melanoma, colorectal and hepatocellular carcinomas. Interestingly, expression of the HCMV-encoded chemokine receptor US28 in intestinal epithelial cells promotes intestinal neoplasia in transgenic mice, which is associated with increased nuclear accumulation of β-catenin. In this study we show that this viral receptor constitutively activates β-catenin and enhances β-catenin-dependent transcription.

Leukocyte ABCA1 remains atheroprotective in splenectomized LDL receptor knockout mice.

Aim: ATP-binding cassette transporter A1 (ABCA1) is an important mediator of macrophage cholesterol efflux. It mediates the efflux of cellular cholesterol to lipid-poor apolipoprotein A-I. LDL receptor (LDLr) knockout (KO) mice deficient for leukocyte ABCA1 (ABCA1 KO→LDLr KO) show increased atherosclerosis and splenic lipid accumulation despite largely attenuated serum cholesterol levels.

TNF-α-mediated NF-κB survival signaling impairment by cisplatin enhances JNK activation allowing synergistic apoptosis of renal proximal tubular cells.

Cisplatin-induced nephrotoxicity is an important limiting factor for cisplatin use. Tumor necrosis factor-α (TNF-α) is known to contribute to cisplatin-induced nephrotoxicity by inducing an inflammatory process aggravating the primary injury, thereby resulting in acute kidney injury (AKI). The present study investigates the pathways synergistically activated by cisplatin and TNF-α responsible for TNF-α-enhanced cisplatin-induced renal cell injury.

Interference of the CD30-CD30L pathway reduces atherosclerosis development.

Objective: Costimulatory molecules tightly control immune responses by providing positive signals that promote T-cell activation or by transducing inhibitory signals that limit T-cell responses. CD30 and CD30L are members of the tumor necrosis factor receptor superfamily and are involved in the activation and proliferation of T and B cells, which have been implicated in the initiation and progression of atherosclerosis. In the present study, we thus aimed to determine the role of the CD30-CD30L pathway in the development of atherosclerosis.