Prostate cancer is a leading killer of men in the industrialized world. Underlying this disease is the aberrant action of the androgen receptor (AR). AR is distinguished from other nuclear receptors in that after hormone binding, it preferentially responds to a specialized set of coactivators bearing aromatic-rich motifs, while responding poorly to coactivators bearing the leucine-rich “NR box” motifs favored by other nuclear receptors. Under normal conditions, interactions with these AR-specific coactivators through aromatic-rich motifs underlie targeted gene transcription. However, during prostate cancer, abnormal association with such coactivators, as well as with coactivators containing canonical leucine-rich motifs, promotes disease progression. To understand the paradox of this unusual selectivity, we have derived a complete set of peptide motifs that interact with AR using phage display. Binding affinities were measured for a selected set of these peptides and their interactions with AR determined by X-ray crystallography. Structures of AR in complex with FxxLF, LxxLL, FxxLW, WxxLF, WxxVW, FxxFF, and FxxYF motifs reveal a changing surface of the AR coactivator binding interface that permits accommodation of both AR-specific aromatic-rich motifs and canonical leucine-rich motifs. Induced fit provides perfect mating of the motifs representing the known family of AR coactivators and suggests a framework for the design of AR coactivator antagonists. A structural study of the androgen receptor shows how it interacts with coactivator proteins. The results also provide a possible framework for the development of new receptor antagonists The androgen receptor (AR) is the cellular mediator of the actions of the hormone 5-α dihydrotestosterone (DHT). Androgen binding to AR leads to activation of genes involved in the development and maintenance of the male reproductive system and other tissues such as bone and muscle. However, it is the pivotal role of AR in the development and progression of prostate cancer that has led to increasing interest in this nuclear receptor. Presently, hormone-dependent prostate cancer is treated with a combination of strategies that reduce circulating levels of androgens, such as the administration of antiandrogens that compete for the androgen-binding pocket in the core of the C-terminal ligand-binding domain (LBD). The benefits of these treatments are typically transient, with later tumor growth associated with increases in expression levels of AR or its cofactors, or mutations that render AR resistant to antiandrogens . AlternaThe precise details of how AR binds the dozens of coregulator proteins reported to associate with different regions of AR in vivo remain poorly understood . Many nuAR, however, utilizes multiple mechanisms to activate gene transcription. Generally, AR activity is dependent on contributions from multiple transactivation functions that lie within the N-terminal domain (NTD) collectively called AF-1. Although the AR AF-2 can bind to a restricted set of LxxLL motifs  and is rPresently, how the AR AF-2 surface can accommodate residues with bulky aromatic side chains and distinguish FxxLF motifs from LxxLL motifs is not known. To understand the structural basis of this unusual coactivator recognition preference, we characterized the full repertoire of interacting sequences using phage display to define amino acids preferred at the AR coactivator binding interface. Crystal structures of the AR LBD in complex with several phage display–derived peptides reveal the structural basis of FxxLF motif specificity and an induced fit of the receptor that allows accommodation of other related hydrophobic motifs. Comparisons of the structures suggest strategies for the design of AR coactivator antagonists.10 randomized peptides against DHT-bound AR LBD. Selections identified sequences containing hydrophobic motifs that were primarily aromatic in character, consistent with another recent study (Phage display has been used to study coactivator recognition specificity and to identify coactivator motif sequence variants preferred by the estrogen receptor (ER), thyroid hormone receptor (TR) β, and most recently AR . Using pnt study    . SurprisP212121 with one molecule per asymmetric unit and unit cell dimensions similar to those observed in previous AR LBD crystal structures  reveals that the AF-2 cleft reorganizes to accommodate the bulky peptide side chains see B and 4. The preference of AR for motifs with aromatic groups over leucine-rich motifs was assessed with a crystal structure of the AR LBD in complex with the LxxLL peptide. The structure reveals similarities between the binding modes of the LxxLL and FxxLF motifs to AR, and other LxxLL motifs to other nuclear receptors. The LxxLL motif adopts a helical conformation, and interactions of the motif with the AF-2 cleft are predominantly hydrophobic, with the three leucine residues of the motif contributing most of the interactions. However, significant differences can be seen between the binding mode of the LxxLL motif to AR and that of p160-derived LxxLL motifs to other nuclear receptors. First, flanking residues were largely disordered, with only two N-terminal flanking residues and one C-terminal residue visible in electron density maps see  and 3B. Side chains of residues flanking the first leucine of the motif make additional hydrophobic interactions with the AR surface see B. Trp+2 To understand how the AR AF-2 accommodates tryptophan residues, structures of AR in complex with peptides containing tryptophan substitutions at the +1 or +5 position, or both, were determined . SurprisFinally, effects of substitutions at the +4 position were assessed in structures of AR in complex with peptides containing FxxFF and FxxYF motifs . SurprisBy contrast, the conformation of FxxYF was the closest to FxxLF see A. Other Together, the structures described above permit an assessment of the way that individual subsites of the AR AF-2 cleft accommodate hydrophobic groups. The indole rings of tryptophan and the phenyl rings of phenylalanine fit into their pockets analogously with the +1 and +5 residues bound facedown and edgewise, respectively, into the AF-2 cleft. On the other hand, the position of the +4 residue is variable, with binding in this shallow pocket largely dictated by the position of the peptide backbone caused by the bound conformations of the +1 and +5 residues see C. Small The binding mode detected in the +1 pocket is the most conserved of the three hydrophobic subsites see C. The beBinding of tryptophans in the +5 pocket is slightly more variable see C. Trp+5 The crystal structures reported here reveal how AR binds coactivator motifs with bulky aromatic hydrophobic groups and permit construction of a profile of the AR coregulator interface see . In someThe same characteristics that make the AR AF-2 ideal for binding of longer, aromatic side chains also make it less well suited for binding of shorter, branched side chains. Although changes in the position of Met734 widen the groove towards the +5 subsite to permit binding of leucine residues, the gross features of the groove remain largely the same see B. As a rUnlike the conserved interaction modes of aromatic residues with the +1 and +5 sites, binding interactions at the +4 site are variable and characterized by nonspecific interactions. This finding agrees with the relatively high conservation of residues at the +1 and +5 positions of AR-interacting motifs and suggests that these residues drive peptide interaction with the LBD, whereas the +4 site is less critical. Indeed, the +4 pocket is shallow, surface exposed, and relatively featureless, explaining the assortment of residues selected at the +4 position. It is likely that any hydrophobic residue that does not clash with surrounding residues would be suitable at this subsite.While peptide motif recognition is governed by hydrophobic interactions, polar interactions from backbone atoms and residues outside the core motif also contribute. With the exception of FxxFF, motifs containing phenylalanines at the +1 and +5 positions present canonical main chain interactions with both charge clamp residues, Lys720 and Glu897. This finding stands in contrast to predictions of previous studies , which cOn the other hand, Glu897 interacts with the FxxLF peptide backbone, but is disengaged from the LxxLL peptide backbone. One possible explanation for the apparent requirement for Glu897 in LxxLL binding is that it might interact with residues outside of the core motif. The corresponding glutamate of GR, Glu 755, forms hydrogen bonds with the −3 asparagine of TIF2 NR box 3 , and GluSequence alignment of NR coactivator sequences shows that positively charged residues are favored N-terminal to the core hydrophobic motif while negatively charged residues are favored C-terminal to the motif . Our phaFinally, the AR AF-2 surface is an attractive target for pharmaceutical design. Selective peptide inhibitors that bind the AF-2 surface of liganded ERα, ERβ, and TRβ have been developed , and simE. coli strain BL21 (DE3) STAR in the presence of 10 μM DHT. Induction was carried out with 30 μM IPTG at 17 °C for 16–18 h. E. coli cells were lysed in buffer  supplemented with 0.5 μg/ml lysozyme, 5 U/ml benzonase, 0.5% CHAPS, and 10 μM DHT. All buffers for further purification steps contained 1 μM DHT. Soluble cell lysate was adsorbed to Glutathione Sepharose 4 Fast Flow resin (Amersham Biosciences), washed with buffer containing 0.1% n-octyl β-glucoside, and eluted with 15 mM glutathione. After cleavage of the GST moiety with thrombin, final purification of the AR LBD was carried out using a HiTrap SP cation exchange column (Amersham Biosciences). Eluted AR LBD was dialyzed overnight at 4 °C against buffer containing 50 mM HEPES (pH 7.2), 10% glycerol, 0.2 mM TCEP, 20 μM DHT, 150 mM Li2SO4, and 0.1% n-octyl β-glucoside, then concentrated to greater than 4 mg/ml for crystallization.Expression and purification of the AR LBD for crystallization were performed essentially as described . The cDNE. coli biotin ligase (Avidity) into BL21 (DE3) STAR cells. Protein expression was carried out as above but with induction supplemented with 50 μM biotin to ensure quantitative biotinylation of AR LBD.Purification of AR LBD for use in phage affinity selection was carried out as above without the final dialysis and concentration steps. The expression construct contained the AR LBD as an inframe fusion with GST in a modified pGEX-2T vector containing both a flexible region and an AviTag sequence  allowing in vivo biotinylation. The GST–AR LBD fusion expression plasmid was cotransformed with a plasmid-encoding 10 different random or biased amino acid sequences were added to the wells containing immobilized AR LBD and incubated for 3 h at 4 °C. After washing, bound phage were eluted using pH 2 glycine. Enrichment of phage displaying target-specific peptides was monitored after each round of affinity selection using an anti-M13 antibody conjugated to horseradish peroxidase in an ELISA–type assay.Phage affinity selections were performed essentially as described . BiotinySynthetic peptides corresponding to the deduced amino acid sequences from receptor-specific phage were tested for their ability to interact with purified AR LBD using a FRET–based assay format. Peptides were synthesized according to the deduced amino acid sequence displayed on phage with an additional C-terminal amino acid sequence consisting of SGSGK to allow the attachment of a biotin tag . Flourophor conjugates were prepared by incubating either biotinylated peptides with streptavidin-cryptate , or biotinylated AR LBD with streptavidin-XL665 (Cis Bio). Interaction between peptide and AR LBD was monitored by the ratio of energy transfer by excitation at 320 nm and emission at 625 nm and 665 nm.Affinities of peptides to the AR LBD were determined with a Biacore  2000 instrument. A peptide derived from silencing mediator for RXR and TR 2 (SMRT2) served as a negative control. 1 mM peptide stock solutions in DMSO were diluted into HBS-P buffer  to generate 10 μM working solutions. HBS-P buffer was flowed through the cells to achieve a stable baseline prior to immobilization of the biotinylated peptides. To achieve the binding of approximately 250 RU of peptides to individual cells, working solutions of peptides were diluted to 100 nM in HBS-P buffer. Unbound streptavidin sites were blocked by injection of a 1 mM biotin solution at a rate of 10 μl/min.Purified AR LBD was diluted into HBS-P buffer to a concentration of 10 μM and injected into all four Flowcells using the Kinject protocol at a flow rate of 10 μl/min . Following the dissociation phase, the surface of the chip was regenerated to remove residual AR LBD by QuickInject of buffer containing 10 mM HEPES and 50% ethylene glycol (pH 11). Following the establishment of a stable baseline, the same procedure was repeated using a series of AR LBD dilutions  in an iterative manner. Analysis of the data was performed using BIAevaluation 3.0 software (Biacore). The SMRT2 signals were subtracted as background from the three remaining peptide signals. Data were best fit using the two-state conformational change model , 2002.Purified, concentrated AR LBD was combined with 3x to 6x molar excess of peptide and incubated 1 h at room temperature before crystallization trials. Complexes were crystallized using the hanging drop vapor diffusion method. Protein–peptide solution was combined in a 1:1 ratio with a well solution consisting of 0.6–0.8 M sodium citrate and 100 mM Tris or HEPES buffer (pH 7–8). Crystals typically appeared after 1–2 d, with maximal size attained within 2 wk. For data collection, crystals were swiped into a cryo-protectant solution consisting of well solution plus 10% glycerol before flash freezing in liquid nitrogen. The addition of ethylene glycol to a well concentration of 10%–20% was later found to both improve crystal quality and enable the freezing of crystals directly out of the drop.A-weighted 2Fo − Fc, Fo − Fc, and simulated annealing composite omit maps. Superposition of structures was performed with LSQMAN , beamline 8.3.1, with either a ADSC Quantum 315 or Quantum 210 CCD detector. Data were processed using Denzo and Scalepack . Moleculh LSQMAN . Buried h LSQMAN . Coordinhttp://www.ebi.ac.uk/swissprot) accession numbers for the gene products discussed in this paper are AR (P10275), ARA54 (Q9UBS8), ARA55 (Q9Y2V5), ARA70 (Q13772), ER , glucocorticoid receptor-interacting protein 1 NR box 3 (Q61026 ), GR (P04150), NR box 3 of TIF2 (Q15596), and TR β (P10828).The Swiss-Prot (http://www.rcsb.org/pdb) accession numbers for the structures used in this paper are FxxFF (1T73), FxxLF (1T7R), FxxLW (1T79), FxxYF (1T7M), LxxLL (1T7F), unbound (1T7T), WxxLF (1T74), and WxxVW (1T76).The Protein Data Bank (


Yukio Mano, MD, PhD (1943–2004)Associate Editor, Journal of NeuroEngineering and Rehabilitation I was terribly shocked to hear of the tragic and sudden passing of Yukio Mano on November 7, 2004, at the age of 61. He had not been well this past year but had been working continuously until just ten days before his death.Yukio Mano Figure  was bornHis research interest was rehabilitation medicine, especially brain plasticity. He was the first Japanese developer of an apparatus that could deliver transcranial magnetic stimulation. Using this apparatus, he analyzed changes in the central nervous system resulting from various diseases, including brain plasticity after anastomosis of the musculocutaneous and intercostal nerves following cervical root avulsion, and cortical reorganization in training. The knowledge resulting from his research proved beneficial in the rehabilitation of disabled patients. He also emphasized a multidisciplinary approach to rehabilitation medicine and adopted new techniques from engineering. He received the Best Paper Award from ANNIE  in 2000 for his work entitled, "Adaptive FES Switching System for Hemiplegics".Yukio Mano served as a council member of the International Society of Electrophysiology and Kinesiology (ISEK) and, in 2000, he organized the XIII Congress of the ISEK in Sapporo, Japan. He was also a member of the editorial board of the Journal of Electromyography and Kinesiology. He served as a council member of many Japanese societies and organized nationwide congresses in Japan even in the year he died. He welcomed the launch of the new Journal of NeuroEngineering and Rehabilitation (JNER) and was honored to be asked to join the Editorial Board as an Associate Editor. He was indeed fully active to his last day.We cannot help praising him for all that he has accomplished in the fields of rehabilitation medicine, neurophysiology and kinesiology. We must also not forget that this remarkable scientist was also a caring family man. He always showed his love for his family as well as for his colleagues and friends. The loss of such an outstanding personality has been met with great sorrow by his family and the international scientific community. We will always remember him with great affection.


Sc. In a cell-based assay, quinacrine inhibits the conversion of normal host prion protein (PrPC) to PrPSc at a half-maximal concentration of 300 nM. While these data suggest that quinacrine may be beneficial in the treatment of prion disease, its penetration into brain tissue has not been extensively studied. If quinacrine penetrates brain tissue in concentrations exceeding that demonstrated for in vitro inhibition of PrPSc, it may be useful in the treatment of prion disease.Prion diseases are caused by the accumulation of an aberrantly folded isoform of the prion protein, designated PrPOral quinacrine at doses of 37.5 mg/kg/D and 75 mg/kg/D was administered to mice for 4 consecutive weeks. Plasma and tissue  samples were taken over 8 weeks: 4 weeks with treatment, and 4 weeks after treatment ended.Sc in cell culture. Particularly extensive distribution was observed in spleen (maximum of 100 μg/g) and liver (maximum of 400 μg/g) tissue.Quinacrine was demonstrated to penetrate rapidly into brain tissue, achieving concentrations up to 1500 ng/g, which is several-fold greater than that demonstrated to inhibit formation of PrPC) to PrPSc will be critical toward its potential benefit.The documented extensive brain tissue penetration is encouraging suggesting quinacrine might be useful in the treatment of prion disease. However, further clarification of the distribution of both intracellular and extracellular unbound quinacrine is needed. The relative importance of free quinacrine in these compartments upon the conversion of normal host prion protein (PrP While the precise site of antiprion action is unknown (intracellular versus extracellular) adequate quinacrine penetration into brain tissue might result in an effective agent for the treatment of prion diseases.Prion diseases, while rare, invariably result in fatal neurodegeneration. At present, no therapy has been proven to be useful in the treatment of prion disease. However, acridine and phenothiazine derivatives have been evaluated ,2 using 0 μg/ml) . HoweverThe objective of this study was to characterize the achievable plasma concentrations and associated tissue  penetration associated with quinacrine.ad lib as a chocolate flavored liquid diet. From weeks 5–8 animals were given regular feed without quinacrine. At weekly intervals, 3 animals of each strain were euthanized and tissues were collected for analysis.The protocol was approved by the institutional animal care and use committee. The Animal Facility of the Institute for Neurodegenerative Diseases provided animal samples from 2 different strains of mice, FVB and CD1. Twenty four animals of each strain  were fed 37.5 mg/kg/D and 24 animals of each strain were fed 75 mg/kg/D of oral quinacrine over a 4-week period (weeks 1–4), given Quinacrine dihydrochloride (purity: 98.6%) was purchased from Fluka, while sulfadimethoxine sodium salt (purity: 98%) which served as the internal standard (IS) was obtained from Sigma. Quinacrine stock solution was prepared as 1 mg/ml in methanol. Sulfadimethoxine stock solution was prepared as 1 mg/ml in 50% methanol. The working solutions were prepared by diluting the respective standard and control stock solutions with 50% methanol and 0.1% formic acid to 2 μg/ml and 100 ng/ml, respectively. All solutions were stored in a 4°C refrigerator in silinized brown glass containers.3OH/H2O/TFA (45:55:0.05) with 1 mM ammonium formate. The flow rate was 0.8 ml/min with 1/4 split into the mass spectrometer. The injection volume was 5–10 μl with a run time of 3.5 min.The HPLC system employed a Shimadzu LC-10 AD pump and a Waters intelligent Sample Processor 717 Plus autosampler/injector. A BDS Hypersil C18 column, 50 × 4.60 mm, 5 μm particle size was directly coupled to a Micromass Quattro LC Ultima triple quadrupole tandem mass spectrometer using electrospray ionization in positive ion mode. The sample cone voltage and collision energy were 25 V and 20 eV respectively for both quinacrine and the internal standard and the source block and desolvation temperature were 100°C and 400°C, respectively. The mass scanning mode employed multiple reaction monitoring (MRM) with the singly charged quinacrine ion selected at m/z 400.5 giving a fragment ion at m/z/142.0, and the internal standard at m/z 311.0→156.0. The mobile phase consisted of CHAll samples were stored at -70°C until analyzed. Each tissue sample was subjected to a specific method, as described below, for drug extraction and for the determination of concentration. All samples were analyzed by LC/MS/MS.Accuracy and precision was demonstrated throughout the working range with interday and intraday coefficient of variation and relative error <10%.Each plasma sample was thawed at room temperature for 10–15 min; then 20 μl of plasma was aliquotted to a new test tube. To each tube 200 μl of 70% acetonitrile solution, containing 0.1% formic acid and 50 ng/mL of internal standard, was added. The test tubes were vortexed at high speed for 1 min and centrifuged at 10,000 rpm for 10 min. The supernatant was transferred into the autosampler for LC/MS/MS analysis. A set of standard curves with a duplicate set of quality control (QC) samples was generated for sample analysis.Prior to the in vivo study, the stability of quinacrine in mouse brain tissue was determined. Using 3 different doses of quinacrine , mouse brain tissue was soaked in 100% methanol for 7 days. Samples were taken on days 0,1,2,3, and 7 to analyze quinacrine concentrations using LC/MS/MS. Complete equilibration was observed by Day 1 and no degradation in quinacrine was noted through day 7.Brain samples were thawed (in plastic tubes) at room temperature for 15–20 min and weighed. To each sample 0.5 ml of 0.9% NaCl was added followed by incubation at room temperature for 1 h. For the standard curve and quality control samples, a brain tissue sample from an untreated mouse was spiked with different amounts of quinacrine and incubated at room temperature for 1 h. After the addition of 100 μl of 1 μg/ml internal standard solution in 50% methanol and 0.1% formic acid, 5 ml of 100% methanol was added into each sample and soaked at 4°C for two weeks. On the last day, 200 μl was aliquotted from each brain sample and placed into the autosampler for analysis via LC/MS/MS.Liver samples were thawed at room temperature for 15–30 min and weighed. Considering the increased size of the liver samples and that the increased connective tissue in liver samples could potentially prevent the complete distribution of quinacrine into methanol, homogenization was used for these samples. To each sample 100% methanol was added (10 ml/g of tissue) and the tissue was homogenized in ice water for 1 min at speed 3 . The internal standard  was added to 0.2 ml of each homogenized liver sample in a glass test tube. Samples were vortexed for 1 min, centrifuged at 3000 rpm for 10 min and 20 μl of each supernatant was transferred into a new test tube. Each sample was further diluted with 4 ml of 50% methanol, vortexed, and 200 μl was transferred to the autosampler for LC/MS/MS analysis. A set of standard curve with a duplicate set of QC samples was generated for sample analysis.Spleen sample preparation was similar to the preparation of brain samples using a methanol soak. Prior to the in vivo study, the stability of quinacrine in mouse spleen tissue was determined. Using 3 different doses of quinacrine , mouse spleen tissue was soaked in 100% methanol for 7 days. Samples were taken on days 0, 1, 2, 3, and 7 to analyze quinacrine concentrations using LC/MS/MS. Complete equilibration took place by Day 1 and no degradation in quinacrine was observed through day 7.Samples from the in vivo analysis were soaked for 14 days at 4°C. On day 14, 50 μl was aliquotted from each spleen sample and diluted with 1 ml of 50% methanol. Each sample was vortexed for 1 min and 200 μl was transferred to the autosampler for LC/MS/MS analysis. A set of standard curves with a duplicate set of QC samples was generated for sample analysis.The mass spectrum of quinacrine Q1) and its tandem spectrum Q3), showing the fragment ion selected for MRM, are shown in Figure  and its , showingIn plasma, quinacrine concentrations with the 37.5 mg/kg/D dose ranged from 75 to 175 ng/ml for both FVB and CD1 strains mice and remained at this level during the 4-week dosing interval. Once discontinued, quinacrine was completely eliminated from plasma, with no drug detectable within one week. At 75 mg/kg/D quinacrine, plasma concentrations reached >2 × those observed with the lower dose, averaging 300 to 400 ng/ml.Quinacrine levels in brain tissue for both mice strains were determined to be substantially greater than those achieved in plasma. Similar to observations with plasma, steady-state levels in the brain with the 37.5 mg/kg/D dose were achieved by the end of the first week, averaging 400 to 600 ng/g brain tissue. Figure th week. Similarly, spleen tissue levels in both mice strains were very elevated, averaging 5 to 10 μg/g with the 37.5 mg/kg/D dose and 40 to 100 μg/g with the 75 mg/kg/D dose. Figure The analysis of liver samples showed particularly high quinacrine levels, at many times those observed in plasma and brain. At the end of the first week of treatment with 37.5 mg/kg/D, steady-state levels of 70 to 90 μg/g were achieved in liver tissue in both mice strains, which remained throughout the course of treatment. Figure Of note, in contrast with plasma and brain tissue, in which dose dependency was somewhat linear, increasing quinacrine doses in spleen and liver were associated with substantially greater tissue levels. In addition to the isolation of quinacrine, numerous metabolites were identified in all tested tissue samples (data not shown).The current study is the first comprehensive evaluation of quinacrine distribution in plasma and brain tissue. Shannon et al. evaluated the pharmacokinetics of quinacrine in the treatment of malaria . Using ain vitro study, the EC50 for quinacrine was 300 nM, which approximates 120 ng/ml. We report here that quinacrine levels in brain tissue averaged 400 to 600 ng/g with the 37.5 mg/kg/D dose and 1500 ng/g with the 75 mg/kg/D dose, concentrations that exceed the in vitro EC50 by 3- to 10-fold.The concentrations we report in brain in terms of concentration/gm of tissue are several-fold greater than that shown for effective antiprion activity in ScN2a cells [in vitro, including clearance of PrPSc (5) and/or inhibition of PrPSc formation [in vitro benefit from quinacrine, found the drug to be ineffective in the animal model under the conditions employed [Others have reported the antiprion function of quinacrine ormation . Barret employed . In contemployed ,8.50, it important to note critical limitations in their interpretation. Quinacrine has been determined to be highly protein-bound [While the current study has confirmed brain tissue concentrations in excess of the reported ECin-bound , measurein-bound . The brain-bound  evaluatein vitro antiprion activity similar to or greater than that of the parent compound. These tests warrant further evaluation.In addition to steady-state levels of quinacrine, we observed a number of quinacrine metabolites in all tissue samples studied, which raises the possibility that these metabolites may have in vitro activity, quinacrine may be a useful agent in the treatment of prion disease. However, more detailed tissue distribution analyses linked with the critical sites of prion action are warranted.Based upon its oral bioavailability, favorable tissue distribution characteristics, and The author(s) declare that they have no competing interests.LY, YH, ETL developed the assay, performed sample analyses, contributed to the writing of the paper. PL, GL, MB, CR developed the protocol and performed the animal model experiments. SBP conceived of the study and participated in its coordination. BJG drafted the manuscript, participated in its coordination and mentored LY. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


On a regional level, our aims were to describe rehabilitation patterns for elderly patients with stroke and hip fracture and to investigate mortality risk during the 6-month post acute period.Data sources included administrative data relative to patients aged 65+ resident in Tuscany admitted in hospital for stroke or hip fracture between 2001 and 2003, traced up to 3 years before and 6 months following index admission. The study design involves computerized linkage of administrative data, and an exploratory analysis of the association between rehabilitation patterns and 6-month mortality, adjusting for clinical, demographic, and acute-related care characteristics using multivariate Cox regression.Rehabilitation patterns vary greatly across Tuscany with considerable cost implications. Six month mortality risk for stroke patients is significantly lower among residents of Local Health Authorities where patients are more frequently rehabilitated, specifically in extra-hospital settings.Our study, targeting two crucial conditions for elderly patients, found a high variability of rehabilitation patterns across a region, albeit coherent between the two pathologies, associated with remarkable differences in average expenditure. Differences in hazard rates for 6-month mortality after stroke at population level were also found. These results need to be confirmed and further investigated through a more robust information framework. National health systems increasingly need to monitor the impact of their policies in order to optimise care. European countries need to identity new solutions for the emerging needs caused by multiple chronic conditions affecting the elderly, frequently as a direct consequence of acute events. Stroke and hip fracture represent major risk factors for the onset of progressive and catastrophic disability  and highBoth conditions typically require a mix of medical/rehabilitative services such as inpatient, outpatient and home-based care, whose integration needs to be carefully monitored . DespiteAims of the study are to describe patterns of utilization of rehabilitation services in Tuscany for subjects aged 65 and older, using stroke and hip fracture as index conditions, and to investigate their association with post-acute 6-month mortality.The study was conducted in the region of Tuscany, Central Italy, with a population of approximately 3.5 million, 23% aged 65+ and 11% over 74. The regional health care system is organized in 12 Local Health Authorities (LHAs) and 4 independent Hospital Trusts (HT). LHAs organize all health services – hospital care included – while HTs provide highly specialized care. According to the latest regional directive in the field of rehabilitation [ad hoc algorithm was used to check the results of the linkage procedure. Residents in one of the 12 LHAs were excluded from the final analysis due to inaccurate recording.The available regional administrative databases  and the regional mortality register were linked through a personal unique identifier (tax file number). An The study population refers to residents in Tuscany aged at least 65, discharged from acute hospital wards between 1/7/2001 and 30/6/2003 with a primary diagnosis of stroke  -16, neveSelected demographic, clinical, acute care-related and ecological rehabilitation variables were identified as potential correlates of mortality. Demographic characteristics included age, gender and marital status. Clinical characteristics included major comorbidities, according to diagnoses available from hospital admissions up to three years prior to the index event -21, typevs the proportion of subjects undergoing inpatient rehabilitation among those rehabilitated (Y axis). A superimposed grid was used to define four quadrants based on the median values of the proportion of rehabilitated patients overall and in hospital: (I) both below the median, (II) overall below the median and in hospital above the median, (III) overall above the median and in hospital below the median, and (IV) both above the median.Univariate statistics and graphical outputs were used to describe rehabilitation services provided after the index event. Percentages are expressed in relation to either the total population experiencing the index event , or, when appropriate, to the subjects discharged alive. We investigated the association between rehabilitation patterns and 6-month mortality through the definition of an ecological variable classifying LHAs by the average use of rehabilitation services for their residents. A specific plot was used to position LHAs  on a bivariate plan showing the proportion of patients rehabilitated after discharge (X axis) rd quartile of LOS in the acute wards. This way we excluded early deaths, both in-hospital and post-discharge not relevant for post-acute rehabilitation, while defining a common start-up for the follow-up of all patients. Multivariate Cox regression analysis was used to evaluate the independent association between rehabilitation and increased mortality, adjusting for all other individual characteristics. Final models were produced forcing in all variables considered clinically meaningful as fixed effects, including individual-level covariates  and an ecological rehabilitation variable [Proportional hazards regression  was the variable . All anavariable , versionth July 2006, Art.3.1.1.b) and is granted direct access to identifiable individual health data (Art.19.1.2).Data processing and statistical analysis have been conducted at the Regional Offices of Agenzia Regionale di Sanità della Toscana. As a technical and scientific structure of the Regional Government, the Agenzia has the right to use administrative databases and publish summary reports  and further 15.0% within 180 days after discharge. For hip fracture, 12,389 subjects were extracted, with 3.1% deaths recorded in hospital (76.7 % of whom before the 3rd quartile LOS) and further 13.8% within 180 days after discharge. Stroke patients were slightly younger than subjects experiencing hip fractures, with a higher prevalence of males. Average LOS was longer among subjects with hip fractures, albeit less variable. In terms of acute care, 549 patients with stroke (4.1%) were admitted to stroke units while 2,801 patients with hip fracture (22.6%) underwent surgery within 2 days from admission. Overall, a lower proportion of stroke patients discharged alive underwent some form of rehabilitation compared to same with hip fracture (25.6 vs 45.5%), but the rehabilitated patients  were similarly distributed in the rehabilitation settings across the two events. Also the median delay between discharge and rehabilitation were similar across the two events, except for inpatient facility care provided to hip fracture patients with a median delay of 4 days vs 2 weeks for stroke. There was no delay for hospital rehabilitation, 15–16 days for home-based care, 35–38 days for outpatient care.Population under study N = 12 LHAs), rehabilitation settings and 6-month mortality rates are described in Figure 2 LHAs, rIn both cases, risk of death during the six-month follow-up was higher for patients who did not undergo any rehabilitation (approx 20%), intermediate for subjects rehabilitated in hospital or at home (between 10–15%), lower for those rehabilitated in outpatient or inpatient settings (below 5%).In stroke, the average economic value of rehabilitation services per rehabilitated subject was 8,860€ for inpatient rehabilitation, 5,025 € for inpatient facility, 489€ for outpatient and 344€ for home-based care. Average economic values for hip fracture were 25% lower for each service, although similarly more expensive for inpatient care than other solutions.Figure rd quartile of the LOS after index admission.Table Adjusted estimates of HRs with 95% CIs are summarized in Table Stroke patients resident in LHAs with a higher proportion of rehabilitated patients  showed a reduced risk of death compared to subjects resident in LHAs with a lower proportion of rehabilitated patients, among whom also the use of in hospital rehabilitation was reduced (quadrant I). However, such reduction was small, not significant  for patients resident in LHAs where most rehabilitation occurs in hospital (quadrant IV), while relatively high and statistically significant  for patients resident in LHAs where most rehabilitation occurs in extra-hospital settings (quadrant III). Among hip fractures, our data did not show any significant association between the average rehabilitation patterns found in LHAs and 6-month mortality.According to our study, approximately 6,500 and 6,000 hospital admissions occur every year in Tuscany for first-ever strokes and hip fracture, matching results obtained using administrative databases ,27 and pThe association found between demographic, clinical and acute care-related factors and 6-months mortality is consistent with recent studies and supports the validity of our data. A large European project in stroke  showed aAs far as acute care is concerned, our results are consistent with a meta-analysis showing a 17% one-year mortality reduction for stroke patients admitted in stroke units , a clearThe mortality risk reduction found for hip fractures promptly operated after admission, inconsistently reported by others ,37, is oOur study highlighted several important aspects related to rehabilitation services.Overall, during the 6-months follow-up period, about 25% stroke and 45% hip fracture survivors used some kind of rehabilitative services. We found a high variation in the use of rehabilitation by LHA of residency, particularly for the fraction of rehabilitation provided in hospital.The patterns of rehabilitation settings are quite consistent across the two acute events, reflecting the different availability of services provided by LHAs across the region, which in turn are the result of long and complex processes rather than of current evidence-based choices.From an economic point of view, the high variability of expenditure found across LHAs raises great concern about equity issues around the provision of rehabilitation services. Similar differences in rehabilitation patterns appear in other European regions and can be very hard to modify.Our results highlight the need for carefully assessing the cost-effectiveness of rehabilitation strategies for patients experiencing stroke and hip fracture. While reducing mortality is not the primary aim of rehabilitation services in general, massive practice of in-hospital rehabilitation was expected to show a protective effect at the population level. In our analysis, subjects with stroke – but not with hip fracture – are instead less likely to die during the six-month post acute period if they live in areas where actually a relatively large proportion of patients undergo rehabilitation services, but more often in extra-hospital settings.The result cannot be fully explained with the available data, so that further investigations are needed to evaluate outcomes of expensive options such as in hospital rehabilitation, for which we still lack a clear evidence of survival improvement at the population level.Finally, several limitations of our study are worth to be outlined.Firstly, the validity of this report is strictly related to the secondary source used. Computerized health records may be still partially incomplete and some of the variation found may be due to the quality of data. To improve quality of this study, we excluded all records relative to one LHA not recording accurately.Secondly, clinical information included in computerized records may not allow an adequate adjustment for the severity of the disease. This may be due partly to incorrect and/or incomplete clinical information, for which we have extended our data collection retrospectively to include all diagnoses available up to three years before the acute event. On the other hand, it can be virtually impossible to isolate the effect of individual characteristics that can be directly related to the health services provided. In our case, at the individual level, the relation between different rehabilitation treatment and 6-month mortality may be confounded by the tendency to offer specific treatment to patients characterized by specific clinical conditions that are in turn associated to the outcome. Consistently with the available literature , we haveThirdly, statistical significance of the association found must be interpreted with caution for methodological reasons. The main results of the study are prone to the "ecological fallacy", i.e. some unmeasured individual characteristics, or other influential ecological confounders may be the actual determinants of the association found between rehabilitation patterns and mortality. To take into account this possible fallacy, we have used all information available related to the individual subject, at the same time providing an interpretation in terms of organizational factors that is highly plausible.Fourthly, economic values are only indirectly estimated, based on the regional tariff lists, which are only a proxy of real health care costs. Furthermore, costs of outpatient services did not include pharmaceuticals. In the present study we have approached the economic analysis only through a rough estimate of the amount of resources spent for rehabilitation, that are certainly underestimated to some extent for the outpatient setting.There is a need for improved local policies for the optimal delivery of health services.Our study, targeting two crucial conditions for elderly patients e.g. stroke and hip fracture, found a high variability of rehabilitation patterns across Tuscany, albeit coherent between the two pathologies, associated with remarkable differences in average expenditure. Differences in hazard rates for 6-month mortality after stroke at population level were also found, with significantly lower rates for those resident in areas where rehabilitation is provided often but in an extra-hospital setting.The methodology presented in this report can be used and further refined to investigate population outcomes while comparing relative strengths and weaknesses of alternative rehabilitation strategies. However, these results need to be confirmed by a more robust information framework, in order to avoid possible ecological fallacyad hoc longitudinal data. More studies are needed to incorporate a range of outcomes properly measured, e.g. the improvement of functional status and the actual costs of health services over multiple diseases.A better assessment of rehabilitation policies requires the construction of composite indicators and the collection of The author(s) declare that they have no competing interests.FC designed the study and statistical analyses, initiating and coordinating the preparation of the manuscript; LR participated in the design of the study and preparation of the manuscript; PF participated in the design of the study and drafting the manuscript; RG participated in the design of the study and performed data transformation and all statistical analyses, developing all STATA software; FT participated in the design of the study and drafted the economic analysis; TDI performed systematic search of the literature; SB performed data extraction and computerized linkage from the regional database; EB conceived, designed and coordinated the study, and participated in drafting the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Docosahexaenoic acid  and n-6 docosapentaenoic acid  are highly unsaturated fatty acids  that differ by a single carbon-carbon double bond at the Δ19 position. Membrane 22:6n-3 may support skeletal muscle function through optimal ion pump activity of sarcoplasmic reticulum and electron transport in the mitochondria. Typically n-3 fatty acid deficient feeding trials utilize linoleic acid  as a comparison group, possibly introducing a lower level of HUFA in addition to n-3 fatty acid deficiency. The use of 22:5n-6 as a dietary control is ideal for determining specific requirements for 22:6n-3 in various physiological processes. The incorporation of dietary 22:5n-6 into rat skeletal muscles has not been demonstrated previously. A one generation, artificial rearing model was utilized to supply 22:6n-3 and/or 22:5n-6 to rats from d2 after birth to adulthood. An n-3 fatty acid deficient, artificial milk with 18:2n-6 was supplemented with 22:6n-3 and/or 22:5n-6 resulting in four artificially reared (AR) dietary groups; AR-LA, AR-DHA, AR-DPAn-6, AR-DHA+DPAn-6. A dam reared group (DAM) was included as an additional control. Animals were sacrificed at 15 wks and soleus, white gastrocnemius and red gastrocnemius muscles were collected for fatty acid analyses.In all muscles of the DAM group, the concentration of 22:5n-6 was significantly lower than 22:6n-3 concentrations. While 22:5n-6 was elevated in the AR-LA group and the AR-DPAn-6 group, 20:4n-6 tended to be higher in the AR-LA muscles and not in the AR-DPAn-6 muscles. The AR-DHA+DPAn-6 had a slight, but non-significant increase in 22:5n-6 content. In the red gastrocnemius of the AR-DPAn-6 group, 22:5n-6 levels (8.1 ± 2.8 wt. %) did not reciprocally replace the 22:6n-3 levels observed in AR-DHA reared rats (12.2 ± 2.3 wt. %) suggesting a specific preference/requirement for 22:6n-3 in red gastrocnemius.Dietary 22:5n-6 is incorporated into skeletal muscles and appears to largely compete with 22:6n-3 for incorporation into lipids. In contrast, 18:2n-6 feeding tends to result in elevations of 20:4n-6 and restrained increases of 22:5n-6. As such, 22:5n-6 dietary comparison groups may be useful in elucidating specific requirements for 22:6n-3 to support optimal health and disease prevention. Docosahexaenoic acid 22:6n-3, DHA) demonstrates health benefits through various physiological systems including neurological , cardiov, DHA demThe impact of dietary fatty acid manipulation on the fatty acid composition of rat skeletal muscle has been studied -10. HoweWhile it is possible to manipulate skeletal muscle 22:6n-3 levels with post-weaning dietary deficiency, it is known that other tissues, specifically those of the central nervous system are resistant to loss of 22:6n-3 with post-weaning deficiency . In addiFeeding 22:5n-6 to achieve reciprocal replacement of 22:6n-3 in muscle may be a novel and ideal control in functional muscle studies. Comparative anatomical studies have demonstrated a link between high frequency contraction and high levels of 22:6n-3 in muscles . Both thThe purpose of the present study is to determine if dietary 22:5n-6 is incorporated into rat skeletal muscle lipids after feeding with a 22:5n-6 oil free of 22:6n-3. The present study uses a unique artificially reared suckling rat model that allowed strict control of the type of fatty acids fed and included: a diet based on 18:2n-6 (AR-LA); and 18:2n-6 diets with either 1% 22:6n-3 (AR-DHA); 1% 22:5n-6 (AR-DPAn-6); or 1% 22:6n-3 plus 0.4% 22:5n-6 (AR-DHA+DPAn-6). A dam reared control group (DAM) was also included. The fatty acid compositions of total lipid extractions were determined for soleus (slow-twitch oxidative), red gastrocnemius (fast-twitch oxidative) and white gastrocnemius (fast-twitch glycolytic) muscles such that a measure of the total lipids in the muscles was obtained. Given the novel artificial rearing approach in the present study and the limited data on dietary 22:5n-6 supplementation, complete fatty acid compositions as detected, are reported rather than selected fatty acids.The body weight of the rats in the present study have been presented previously . BrieflyFatty acid compositions of total lipid extracts from soleus, red gastrocnemius and white gastrocnemius differed in relative weight percentages and concentrations in the dam-reared rats Table . Total fThe fatty acid composition also varied at the level of individual fatty acids Table . AlthougThere were no significant differences in the total concentration of all fatty acids with dietary treatment of each muscle; therefore comparisons of dietary treatments are restricted to relative weight percentages of total fatty acids. The percentage of 22:5n-6 was significantly higher in the AR-LA and AR-DPAn-6 groups in all muscles as compared to rats with dietary 22:6n-3, including the AR-DHA+DPAn-6 group. In fact, the percentage of 22:5n-6 in the AR-DHA+DPAn-6 group was not statistically different than either the DAM or the AR-DHA groups for all muscles. The potential ability of 22:5n-6 to replace 22:6n-3 in skeletal muscle was investigated by comparing the percentage of 22:5n-6 in the AR-DPAn-6 and AR-LA groups to the percentage of 22:6n-3 in the AR-DHA in each muscle. In soleus and white gastrocnemius, there were no differences, however in the red gastrocnemius, the amount of 22:5n-6 in the AR-DPAn-6 (8.1 ± 2.8 wt. %) and AR-LA (5.9 ± 1.9 wt. %) groups were significantly lower than the amount of 22:6n-3 in the AR-DHA group (12.2 ± 2.3 wt. %). While total HUFA was not significantly different in any of the muscles, the sum of 22:6n-3 + 22:5n-6 was significantly lower in the red gastrocnemius of the AR-DPAn-6 (9.2 ± 3.0 wt. %) and AR-LA (6.8 ± 2.1 wt. %) groups as compared to the AR-DHA group (13.5 ± 2.6 wt. %).P < 0.10 for all individual comparisons . Interestingly, the percentage of 20:4n-6 was the highest numerically, but not statistically different in the AR-LA groups of the red gastrocnemius and soleus. The percentage of 18:2n-6 was significantly higher in all muscles in the DAM group as compared to the AR rats. In white gastrocnemius, the AR-DPAn-6, AR-DHA+DPAn-6 and AR-DHA groups also had percentages of 18:2n-6 that were significantly lower than the AR-LA group. The total n-6 PUFA percentage in muscle tended to be lower in the AR-DHA+DPAn-6 and AR-DHA groups and higher in the AR-LA and AR-DPAn-6 groups, mainly as a result of the higher percentage of 22:5n-6. There was an exception in soleus as the highest observed percentage of total n-6 PUFA was in the DAM group because of a significantly higher percentage of 18:2n-6.In the white gastrocnemius, the percentage of 20:4n-6 was significantly higher in the AR-LA group as compared with the AR-DHA+DPAn-6 group, with a tendency for a difference from the other groups as Total PUFA did not differ in red gastrocnemius and white gastrocnemius, but was significantly lower in soleus in the artificially reared as compared to the dam-reared rats Tables , 4, 5. TTotal n-3 PUFA percentages were significantly lower in the AR-LA and AR-DPAn-6 groups and similar for the DAM group and the AR-DHA group in all muscles. In white gastrocnemius and soleus, the total n-3 PUFA percentage in the AR-DHA+DPAn-6 group was significantly lower than the DAM group, but not the AR-DHA group. The AR-LA and AR-DPAn-6 groups had significantly lower percentages of n-3 HUFA in total HUFA than the other groups in all muscles. The response of this parameter to the inclusion of 22:6n-3 in the diet was not homogeneous for all three muscles. In soleus, there were no differences between the DAM, AR-DHA+DPAn-6 and AR-DHA groups. In red gastrocnemius, the DAM group (55.8 ± 1.8%) and AR-DHA group (52.4 ± 3.0%) were similar, but the AR-DHA+DPAn-6 group (47.7 ± 3.3%) had a significantly lower percentage of n-3 HUFA in total HUFA. In white gastrocnemius, the DAM, AR-DHA and AR-DHA+DPAn-6 groups were statistically different for each muscle at 54.2 ± 2.0%, 48.6 ± 3.6% and 43.8 ± 4.1%, respectively.There were also differences observed in the saturated and monounsaturated fatty acids. Total saturates were significantly lower in all muscles of the AR rats as compared to the DAM group. This was largely driven by higher percentages in 12:0, 14:0, 20:0, 24:0 with a tendency for higher percentages of 18:0 and 22:0 in the DAM group muscles, while the percentage of 16:0 was largely similar. Total percentage of monounsaturates was lower in the DAM group, although the difference was not statistically different in the red gastrocnemius. This was mainly a result of significantly higher 18:1n-9 in all muscles.The present study identifies a novel research method that can assist in determining the requirement of 22:6n-3 for proper skeletal muscle function including insulin resistance and diabetes. This study also confirms that dietary fatty acid intake can influence the fatty acid composition of muscle ,10,26-28In the present study, the percentage of total HUFA was not affected by the dietary manipulations although the amount of HUFA appears to be specific to each muscle type. There were obvious differences in the percentage of n-3 HUFA in total HUFA, as the diets without 22:6n-3 had markedly lower n-3 HUFA. The AR-LA group, which had no preformed dietary HUFA, did not differ however in total HUFA, and had the lowest percentage of n-3 HUFA and the highest 20:4n-6 wt. % in each muscle . Calculations for 80% power indicate that to determine significant differences  between the percentage of 20:4n-6 in the AR-LA group and the AR-DPAn-6 group, n = 17, n = 31 and n = 53 would be required for each group for white gastrocnemius, red gastrocnemius and soleus, respectively. Although highly speculative, it appears that the AR-LA group may have had higher rates of elongation and desaturation of 18:2n-6 to 20:4n-6. Fatty acid isotope studies are required to verify this hypothesis.The observation that 22:6n-3 levels are low in muscles when it is absent in the diet was expected. The 22:6n-3 levels presently observed in the AR-LA group  are in the same range as the 1.5 wt. % in phosphatidylethanolamine, 0.84 wt. % in phosphatidylserine + phosphatidylinositol and 0.14 wt. % in phosphatidylcholine reported by Tinoco et al.  and lowesn-2 position of phospholipids. HUFA incorporation into neutral lipids such as triacylglycerols is often minor and undetectable as demonstrated recently in the rat [Both 22:5n-6 and 22:6n-3 are HUFAs that are found predominantly in, and potentially compete for the  the rat . The fat the rat  with red the rat ,24. ThesThe lipid class composition greatly influences the overall fatty acid composition of a tissue. In addition, the proportion of muscle membranes; the sarcolemma, sarcoplasmic reticulum and mitochondria, differ between muscle fibre types ,30. MixeThe sarcolemma is unique in that the neutral lipid content is approximately equivalent to the phospholipid content and that neutral lipid composition approaches an even distribution between triacylglycerols, non-esterified fatty acids, cholesterol and cholesterol esters. In both the sarcoplasmic reticulum and mitochondria, phospholipids consist of approximately 80% of total lipids with triacylglycerol being the dominant neutral lipid and both fractions have similarly high percentages of 22:6n-3 (approximately 20%) . HoweverAlthough exercise training has also been demonstrated to result in changes in fatty acid composition ,8,35,36 Overall, the present study demonstrates that animals fed 22:5n-6 could be an important comparison group when examining the physiological impact of 22:6n-3. Feeding 22:5n-6 in comparison to feeding 22:6n-3 results in fatty acid composition changes that are largely restricted to 22:5n-6 and 22:6n-3 fatty acids. Feeding with 18:2n-6 feeding appears to increase 20:4n-6. This study also demonstrates that artificial rearing together with the use of semi-synthetic milk substitute is an effective method to manipulate skeletal muscle fatty acid compositions and to induce 22:6n-3 deficiency. The present study also demonstrates that muscle fibre type discrimination is important and that 22:6n-3 requirements may differ between fibre types, possibly in relation to sarcoplasmic reticulum and mitochondrial oxidation functions. It should be noted that the high percentage of 22:6n-3 observed in the in red gastrocnemius of the DAM group is comparable to levels found in brain in which 22:6n-3 has been demonstrated to have a critical role in supporting optimal function. The high level of 22:6n-3 in these muscles is believed to be a component of phospholipids that support optimal functioning of ion pump activity of sarcoplasmic reticulum and electron transport in the mitochondria as docosahexaenoic acid supports optimal G protein-coupled receptor signalling in retinal rod outer segments . The defad libitum water and at a controlled temperature (23 ± 1°C) and a 12-hr light/dark cycle. At postnatal day 2, male pups were collected from each litter and randomized to one of five experimental groups. The groups included a dam-reared group and groups artificially reared on one of four experimental milks. The composition of the experimental milks was based on an artificial milk with fat content from hydrogenated coconut oil , medium chain triglycerides  and oleic and linoleic ethyl esters with purified docosapentaenoic n-6 and/or docosahexaenoic ethyl esters added . Thus, the five treatments included a dam-reared diet (DAM) where the dams received an n-3 fatty acid adequate  but 22:6n-3 free diet, and four artificially reared (AR) groups. The four AR groups received the following diets: a 15% of total fatty acid 18:2n-6 based diet (AR-LA), the 18:2n-6 based diet with 1% of total fatty acids 22:5n-6 (AR-DPAn-6), the LA based diet with 1% of total fatty acids 22:6n-3 (AR-DHA) and a 18:2n-6 based diet with 1% of total fatty acids 22:6n-3 and 0.4% of total fatty acids 22:5n-6 diet (AR-DHA+DPAn-6). Details on the composition of the diets have been presented previously [ad libitum from water bottle nipples after eye opening  and were eventually weaned to a pelleted diet with a similar fatty acid composition with the DAM group transferred to the maternal diet. The male pups underwent a series of behavioral assessments as described previously [A detailed description of the study design and experimental diets has been published previously . All exp3 in methanol according to Morrison and Smith [Total lipids were extracted from muscles by a modified Folch procedure  with docnd Smith  with a mnd Smith . Fatty and Smith .t test comparisons of 22:5n-6 in the AR-DPAn-6 group with 22:6n-3 in the AR-DHA group.All statistical analyses were completed with SPSS for Windows statistical software . All data is expressed as mean ± standard deviation (SD). Comparisons between muscle types of dam-reared rats were completed by repeated measures ANOVA with each animal (n = 6) providing a complete set of the three muscle tissues. The effects of dietary treatment were compared by the General Linear Model procedure. Individual means were compared using Tukey's honestly significant difference test after a significant F-value was determined. Statistical significance was inferred at P < 0.05. The potential for dietary docosapentaenoic acid to replace docosahexaenoic acid was examined by The author(s) declare that they have no competing interests.KDS contributed to the study design, assisted in animal rearing, performed muscle collection and fatty acid analyses and statistical analyses and was the primary author. SYL contributed to the study design, managed animal rearing, and contributed to manuscript revision. NS contributed to the study design, coordinated the study design and contributed to manuscript revision. All authors read and approved the final manuscript.


The presence of A2A and A2B ARs was assessed by immunoblotting using specific antibodies. Equilibrium A2A and A2B ARs binding parameters were evaluated by radioligand binding assay. Functional studies were conducted to investigate coupling of the A2B AR to the adenylyl cyclase pathway. This is the first report of the use of Western blot analysis to confirm the presence of A2A and A2B ARs in human neutrophils. No significant changes in A2A AR binding parameters or expression levels were detected between SSc patients and healthy control individuals. A significant decrease (65%) in the maximum density of A2B AR binding sites occurred in SSc neutrophils, whereas no changes in the affinity constant values were found. Moreover, a decrease in A2B AR mediated adenylyl cyclase activity was observed in patients with SSc. Our findings demonstrate the occurrence of selective alterations in A2B AR density and signalling in SSc.We conducted the present study to investigate protein expression and functioning of A Systemic sclerosis (SSc), also known as scleroderma, is a connective tissue disease of unknown aetiology. Possibly an autoimmune disorder, it is accompanied in the vast majority of cases by the presence of antinuclear antibodies . SSc mayThe mechanisms leading to endothelial damage, inflammation and fibrosis are unclear. Reactive oxygen species in neutrophils may increase the extent of inflammation and fibrosis during the respiratory burst and could be involved in endothelial damage . The end1, A2A, A2B and A3 have been identified and their functions characterized in neutrophils. Specifically, activation of A1 ARs enhances chemotaxis, phagocytosis and adherence CGS21680 (CGS21680 = [2-p-(2-carbowyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine), [3H]NECA (NECA = 5'-N-ethylcarboxamidoadenosine), and [32P]α-ATP were supplied by NEN Life Sciences . Electrophoresis reagents were purchased from BioRad . A2AAR and A2BAR antibodies were supplied by Alpha Diagnostic . All other chemicals were from standard commercial sources.Bacitracine, benzamidine, trypsin inhibitor, sodium orthovanadate, Nonidet P-40, SDS, phenylsulfonylfluoride, aprotinin and adenosine deaminase (ADA) were purchased from Sigma . Unlabelled AR agonists/antagonists and the anti-β-actin antibody were supplied by RBI/Sigma . CGS21680. Aliquots of neutrophil membranes (0.2–0.3 mg protein) were incubated with different [3H]CGS21680 concentrations (5–30 nmol/l) in a final volume of 250 μl of T2 buffer. Nonspecific binding was determined in the presence of 100 μmol/l NECA. After 90 min incubation at 25°C, the binding reaction was terminated by vacuum filtration through Whatman GF/C glass fibre filters , accompanied by three washes with ice-cold T2 buffer (4 ml). A2A AR specificity was evaluated through competition experiments, using different AR ligands.A2B AR binding assay was performed using 20 nmol/l [3H]NECA in the presence of 50 nmol/l cyclopentyladenosine (CPA) and 100 nmol/l SCH58261  to prevent [3H]NECA binding to A1 and A2A ARs, respectively cAMP  to monitor column recovery. Newly formed ZnCO3 allowed precipitation of residual ATP, discarded through centrifugation at 2700 g for 8 min. Supernatants containing both [32P]α-cAMP and [3H]cAMP were further purified by double-step Dowex-Alumina chromatography and counted by means of a β-counter .Neutrophils were homogenized in buffer solution containing 10 mmol/l Hepes, 1 mmol/l EGTA and 10 mmol/l NaCl Salomon  and John Salomon , with so2B AR mediated cAMP accumulation, the reaction was carried out in the presence of selective A2A antagonist SCH58261 at a concentration (100 nmol/l) able to block A2A receptors completely [To evaluate Ampletely .max) were calculated using the nonlinear multipurpose curve-fitting computer program Graph-Pad Prism The 50% inhibitory concentration values were calculated using the same program and converted to Ki values through the Cheng and Prusoff equation.Affinity constant values (Kd) and maximum number of binding sites (BP < 0.01) was used to determine binding data with the best fit to a one-site or two-site model. Differences in binding parameters between SSc patients and control individuals were evaluated by one-way analysis of variance.A GS-670-BIO-RAD imaging densitometer was used for semiquantitative analysis of immunoblots. Partial F test . Scatchard analysis for SSc neutrophils revealed no significant differences in Kd and Bmax between patients  and healthy control individuals .The selective A2B AR binding sites were identified using [3H]NECA as radioligand in the presence of 50 nmol/l CPA and 100 nmol/l SCH58261, to prevent nonspecific binding to A1 and A2A AR subtypes. We performed competition experiments using a wide range (50 nmol/l to 2 mmol/l) of [3H]NECA concentrations to allow the identification of A2B AR low-affinity binding sites. Data analysis revealed that the one-site model produced a significantly better fit than the two-site model (P < 0.05), both in control and SSc neutrophils. In our experimental conditions, control neutrophils exhibited the presence of low-affinity binding sites with Kd and Bmax values of 476 ± 34 nmol/l and 3696 ± 210 fmol/mg, respectively  as compared with controls  on AC activity. NECA stimulated AC activity in a concentration dependent manner. Dose-response curves revealed significant differences between SSc patients  and controls , suggesting an alteration in A2B AR responsiveness in SSc  was observed, with no significant change in Kd values.A2B ARs are known to be low-affinity adenosine binding sites. Competition experiments using a variety of A2B AR agonists and antagonists revealed a pharmacological profile typical of A2B ARs, which is consistent with studies conducted in transfected cell models. Our findings represent the first characterization of A2B ARs in neutrophils with binding experiments.A2 ARs, SSc patients were randomly divided into two subgroups. No difference was found when the two groups were compared, suggesting that different degrees of disease severity and activity had no impact on the assays, but that the disease per se is required to modulate levels and functioning of A2B receptors.In order to analyze a population of nonhomogenous patients and to evaluate the impact of the disease on A2B ARs was accompanied by alterations in receptor responsiveness. An evaluation of the ability of NECA to increase AC activity revealed functional coupling of A2B receptors to G proteins. In SSc patients a significant reduction (by more than 50%) in NECA potency was observed, without any effect on agonist efficacy.Functional studies were performed to investigate whether the decrease in level of A2B AR levels and responsiveness occurred in SSc. Alterations in the expression and functionality of A2B ARs (low-affinity ARs) in patients with SSc may be responsible for the increase in free oxygen radicals, and consequent oxidative damage, that characterizes SSc. This would account for impaired control of hypoxic and inflammatory processes.Our findings suggest that a selective reduction in A2 - generation, phagocytosis and cell adherence by occupying specific A2 ARs. Because hypoxia, ischaemia and inflammation can stimulate adenosine production, A2 AR regulation has been postulated to be a self-protective mechanism for cells from activated neutrophils pheylethylamino)-5'N-ethylcarboxamidoadenosine; CPA = cyclopentyladenosine; Kd = affinity constant; NECA = 5'-N-ethylcarboxamidoadenosine; R-PIA = R-N6-phenylisopropyladenosine; SCH58261 = 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo-1,2,4-triazolopyrimidine; SSc = systemic sclerosis.AC = adenylyl cyclase; ADA = adenosine deaminase; AR = adenosine receptor; BThe author(s) declare that they have no competing interests.LB organized the study design and recruited the patients. LT carried out the binding experiments and statistical analysis. AR participated in the immunoblotting experiments and helped to draft the manuscript. FdF participated in the collection of human samples. AL participated in the coordination of the study and helped with problem solving. SB participated in the coordination of the study and in planning the manuscript. CM participated in the coordination of the study and designed the AC assay. All authors read and approved the final manuscript.


In pancreatic beta cells, sulfatide has been shown to be involved in insulin processing and secretion in vitro. In this study, we examined the uptake of exogenously administered sulfatide and its distribution to the pancreatic beta cells. This might encourage future studies of the function(s) of sulfatide in beta cell physiology in vivo. Radioactive sulfatide was given orally to mice whereafter the uptake of sulfatide in the gastrointestinal tract and subsequent delivery to the pancreas was examined. Sulfatide uptake in pancreas was also studied in vivo by i.p. administration of radioactive sulfatide in mice, and in vitro in isolated rat islets. Isolated tissue/islets were analysed by scintillation counting, autoradiography and thin-layer chromatography-ELISA.The glycosphingolipid sulfatide has previously been found in several mammalian tissues, but information on the uptake of exogenously administered sulfatide in different organs in vitro studies in isolated rat islets support that sulfatide, independently of its fatty acid length, is endocytosed and metabolised by pancreatic islets.Sulfatide was taken up in the gastrointestinal tract for degradation or further transport to other organs. A selective uptake of short chain and/or hydroxylated sulfatide fatty acid isoforms was observed in the small intestine. Exogenously administered sulfatide was found in pancreas after i.p, but not after oral administration. The Our study supports a selective uptake and/or preservation of sulfatide in the gastrointestinal tract after oral administration and with emphasises on pancreatic sulfatide uptake, i.p. administration results in sulfatide at relevant location. Severin vitro -12 and iin vitro [The sulfatide fatty acid isoform varies between cell types and tissue structures. In the brain, which has a high sulfatide content, the major source is myelin where sulfatide mainly contains long unsaturated and hydroxylated fatty acids. In the pancreatic beta cells, saturated fatty acids without hydroxylation are major isoforms. The short 16 carbon atom (C16:0) isoform comprises one and the longer 24 carbon atom (C24:0) the other major species in islets ,13. The Type 2 diabetes patients are characterised by insulin resistance and impaired insulin secretion, and animal models are commonly used for pathophysiological studies of this disease. The beta cell related C16:0 sulfatide isoform was found in reduced amounts in the type 2 diabetes animal models ob/ob and db/db mice, as compared with non-diabetic Lewis rats, BALB/c mice and human pancreatic tissue . Furtherin vivo and its potential pharmacological effects, it is of relevance to explore the uptake of this molecule in pancreas and islet cells. Concerning oral administration, there have been few studies of intestinal digestion, uptake and further organ distribution of glycosphingolipids, including sulfatide. Among these are investigations by Nilsson et al., [et al. [In anticipation of further studies of the role of sulfatide in beta cell physiology  et al., ,21, more [et al. , thus re [et al. ,24, and i) uptake in the gastrointestinal tract and further transport to pancreas was studied by oral administration of radioactive sulfatide to BALB/c mice, ii) uptake of specific sulfatide isoforms (radioactive) by the pancreas after i.p injections in the type 2 diabetic model ob/ob mouse, iii) sulfatide uptake and metabolism, in vitro, in isolated islets of Langerhans prepared from Lewis rat was examined and different isoforms of sulfatide were compared.To explore the uptake of exogenously added sulfatide, the following experiments were performed: 3H-sulfatide taken up in the gastrointestinal tract was determined by scintillation counting, and radioactivity was found in isolated stomach, duodenum/jejunum, ileum and terminal ileum 1–6 hours after administration . The presence of two sulfatide bands in the administered sulfatide fraction  was measured using TLC-ELISA in the respective tissues, and the results are summarized in Table 3H-sulfatide isoforms (C12:0 and C16:0) taken up by pancreas 6 or 24 hours after i.p injection were determined using scintillation counting. In animals injected with C12:0 sulfatide, 1202 ± 275 dpm/mg protein (n = 4) and 1520 ± 442 dpm/mg protein (n = 4) were detected 6 and 24 hours after administration, respectively. When C16:0 sulfatide were administered, 789 ± 135 dpm/mg protein (n = 6) and 656 ± 93 dpm/mg protein (n = 6) were detected 6 and 24 hours after administration, respectively. There were no significant changes in radioactivity (cpm/mg protein) when the different isoforms or the two time periods were compared. Approximately 0.5-1% of the radioactive labeled sulfatide isoforms given to the animals were found in pancreas after 6 and 24 hours, corresponding to approximately 1 nmol sulfatide, assuming that the sulfatide molecule was still intact.Radioactive 3H-sulfatide was present in isolated pancreas, saponified sulfatide fractions from pancreatic samples were analysed using autoradiography and compared with structurally characterized sulfatide isoforms (C12:0 and C16:0 sulfatide). The results show that both C12:0 and C16:0 sulfatide isoforms are taken up by the pancreas  in the pancreatic tissue of ob/ob mice was examined using TLC-ELISA. Mice receiving C16:0 sulfatide showed significantly lower pancreatic sulfatide concentrations 24 hours after administration  than 6 hours after sulfatide administration . Thus, these results lent support to the idea of sulfatide being degraded 24 hours after administration. Further support for an uptake of C16:0 sulfatide in pancreas 6 hours after i.p administration is the occurrence of a slow migrating band on the TLC plate Figure , lane 7,Owing to the fact that the Sulph I antibody has a 5–10 fold lower affinity for C12-C14 sulfatide isoforms compared with C16-C24 isoforms (unpublished data), we were unable to obtain reliable data regarding the total amount of sulfatide (endogenous pool plus exogenous uptake) in the animals receiving C12:0 sulfatide.in vitro , which is considered a relatively large amount of sulfatide, since 500 Lewis rat islets normally contains approximately 1–2 nmol sulfatide [The C16:0 isoform of sulfatide was shown to be taken up by the islets of Langerhans tro Fig. , n = 4. tro Fig. . Part oftro Fig. , probabl3H-C12:0 sulfatide and 1.9 ± 0.6%  of 3H-C24:0 sulfatide were taken up by the islets after 6–24 hours, corresponding to 0.3 ± 0.1 nmol  and 1.0 ± 0.3 nmol , respectively.The C12:0 and C24:0 sulfatide isoforms were also taken up by the rat islets, but since a lower amount of radioactivity was incorporated in these sulfatide isoforms (dpm/nmol sulfatide), as compared with C16:0 sulfatide, the degradation products could not be visualized on the TLC-plate Fig. , Table 3in vitro studies further show the ability of sulfatide to be endocytosed and metabolized by pancreatic islet cells. Sulfatide can also be taken up in the gastrointestinal tract after oral administration, which includes uptake and/or preservation of selected fatty acid isoforms of sulfatide. However, with the experimental conditions used in this study, oral administration of sulfatide did not result in a distribution to the pancreatic islet cells.This study supports the idea that sulfatide can be taken up by pancreas when administered i.p, and et al. [The presence of sulfatide in the gastrointestinal tract of mammals has been reported in several studies ,25-27 anet al. ,27 suggeet al. . The banOral administration of the sphingolipid sphingomyelin and simple glycosphingolipids as galactosylceramide/glucosylceramide, has shown that these compounds undergo very little cleavage in the stomach, but are instead hydrolyzed in the small intestine of mammals by alkaline sphingomyelinase, glucosylceramidase and ceramidase ,22,29,30Although our data suggest that oral administration of sulfatide does not result in further distribution to pancreas, uptake of sulfatide in the intestine might still be of interest in relation to its role in type 1 diabetes (an autoimmune disorder) for a number of reasons: 1) Sulfatide antibodies is present in type 1 diabetic individuals ,32, indiin vitro experiments using isolated rat islets. Thus the radioactivity remaining in pancreas 24 hours after administration probably represents intact sulfatide plus the degradation product(s) of this molecule.Since ob/ob mice selectively lack the C16:0 sulfatide isoform in pancreas , this moin vitro studies further support the C12:0 and C16:0 isoforms being taken up by islets, and this was also observed with the long chain beta cell sulfatide isoform (C24:0). The physical/chemical properties of sulfatide vary with different fatty acid length, including their ability to be incorporated in lipid membranes. In the present study, this phenomenon is evident in the intestine, where selected isoforms were taken up, but seem not to be the case for pancreatic islets. Thus, our results suggest that the uptake of different sulfatide isoforms in islets occurs independently of the length of the fatty acid chain, i.e. an unspecific endocytosis occurs. Sulfatide is further degraded within the islets and the production of ceramide observed probably represents a degradation product of sulfatide within the lysosomes, which is unable to exit this compartment [Both non-native C12:0 sulfatide and the C16:0 isoform were given i.p, and similar results regarding pancreatic uptake was observed. The partment . This fipartment .in vivo experiments exploring the role of sulfatide in beta cell function and pathogenesis of type 2 as well as type 1 diabetes.In conclusion, i.p administration of the glycosphingolipid sulfatide results in uptake in pancreas, which probably represents endocytosis by pancreatic beta cells. Although oral administration did not show pancreatic uptake, sulfatide was taken up in the gastrointestinal tract, where specific fatty acid isoforms of sulfatide are preserved. These results suggest that sulfatide could be endocytosed in relevant compartments for future ob (ob/ob) mice were purchased from Bomholtgaard . The principles of laboratory animal care , were followed, and the experiments were approved by the Danish council for animal welfare under the Ministry of Justice.Lewis rats  were purchased from Møllegaard  and BALB/c mice  from Bomholtgaard  and bred in the stables at Bartholin Instituttet, Copenhagen, Denmark. Male, C57BL/6J-LepSulfatide and galactosylceramide used as standard were purified from pig brain according to the procedure previously described . Other lThe synthesis of sulfatide with uniform fatty acids has previously been described . Briefly2/NaB3H4, as previously described [Structurally characterized sulfatide, isolated from brain or synthesized with a uniform fatty acid, galactosylceramide and ceramide, used as standards, was tritium labelled in the ceramide portion with Pd(OAc)escribed .3H-sulfatide giving the specific activity of 15 000–70 000 dpm/nmol sulfatide, and the mixture was sonicated with a Branson S-250 sonifier  for 2 × 20 sec at 25 W prior to use. The concentration of 3H-sulfatide in the solution was determined by scintillation counting, and >50% was found in solution.Phosphate buffered saline (pH 7.2) was added to tubes containing evaporated sulfatide derived from brain , including 6 dpm/mouse). The animals were sacrificed (by carbon dioxide asphyxiation) after 1–2 hours (n = 8), 5–6 hours (n = 7) or 24 hours (n = 7). Food and water were allowed ad libitum. From each animal, stomach, intestine, liver, pancreas and blood were dissected under sterile conditions. The intestine was divided into three parts, representing duodenum/jejunum, ileum and terminal ileum and these three parts were rinsed with approximately 2 mL phosphate buffered saline after dissection. Serum was derived from the blood samples for analysis. Tissue samples were stored at -20°C prior to analysis.Male, 8–10 weeks old BALB/c mice were divided into three groups and given brain derived sulfatide orally  for 2 × 20 sec at 25 W prior to use. The recovery of 3H-sulfatide in the solution was determined by scintillation counting and approximately 90% of C16:0 and C12:0 sulfatide were found in solution.Phosphate buffered saline (pH 7.2) was added to tubes containing evaporated sulfatide with a specific fatty acid length (500 nmol/mL of C12:0 and C16:0 sulfatide), including 6 dpm/mouse, 135 nmol/mouse). The animals were sacrificed (using carbon dioxide asphyxiation) after 6 hours  or 24 hours . Food and water were allowed ad libitum. From each animal, pancreas was isolated under sterile conditions and tissue samples were stored at -20°C prior to analysis.Male, 8–10 weeks old, ob/ob mice (blood glucose 5–15 mM) were divided into two groups and given C12:0 or C16:0 sulfatide i.p  by sonication  for 2 × 20 sec at 25 W prior to use. The recovery of 2 at 37°C. During this period of culturing, heat inactivated fetal calf serum was used (56°C for 45 minutes). Islets were harvested and washed in phosphate buffered saline by centrifugation and stored at -20°C prior to analysis.Islets were divided into 500 islets/culture dish, put into culturing media (as described above) containing the separate sulfatide isoforms and cultured for 6 hours (n = 4) or 24 hours (n = 4) in an atmosphere of 95% air-5% CO3H-sulfatide taken up by the tissue. Approximately 50 μL of the serum samples were analysed by scintillation counting. Further purification was performed by silica gel-60 chromatography , as previously described [Lipids were extracted from homogenates of tissues/islets, after protein determination , by adding methanol and chloroform to give a final ratio of chloroform/methanol/water  . An aliqescribed , where sAliquots of the saponified "sulfatide fractions", corresponding to 2 500–3 500 dpm (tissue), 3 500–8 500 dpm (islets cultured with C12:0 and C24:0 sulfatide) or 35 000–100 000 dpm (islets cultured with C16:0 sulfatide) were applied as 8 mm bands to alumina-backed high performance-TLC plates (Merck) and chromatographed in C/M/W . The plate was air dried, sprayed with Enhance spray  and exposed to x-ray film for 7–14 days. The individual sulfatide bands after chromatography were identified by comparing with the migration of structurally identified sulfatide standard(s). Autoradiography performed on the "sulfatide fraction" purified from islets also included structurally identified standards of galactosylceramide, sphingomyelin and ceramide. In this experiment, liquid scintillation counting, as described above, was performed on samples after saponification and on scraped out fractions of the individual lipid bands after chromatography.Sulfatide was identified and quantified using a TLC-ELISA method previously described  using thThe sulfatide content and/or uptake in tissues and islets were presented as mean values ± SEM. The significance of difference was evaluated by using Student's t-test (significance level set at < 0.05).The author(s) declare that they have no competing interests.MB carried out part of the lipid analyses and collected the data, participated in the design of the experiments and drafted the manuscript. TO carried out the administration of sulfatide to mice. J-E M and KB participated in interpretation of data and were involved in revising the manuscript critically. PF made substantial contribution to the design of the study, interpretation of data and in revising the manuscript critically. All authors read and approved the final manuscript.


The androgen receptor (AR) plays critical roles in both androgen-dependent and castrate-resistant prostate cancer (PCa). However, little is known about AR target genes that mediate the receptor's roles in disease progression.in vivo AR-mediated regulation of several genes identified by ChIP Display. For example, PRKCD and PYCR1, which may contribute to PCa cell growth and survival, are expressed in PCa biopsies from primary tumors before and after ablation and in metastatic lesions in a manner consistent with AR-mediated stimulation.Using Chromatin Immunoprecipitation (ChIP) Display, we discovered 19 novel loci occupied by the AR in castrate resistant C4-2B PCa cells. Only four of the 19 AR-occupied regions were within 10-kb 5'-flanking regulatory sequences. Three were located up to 4-kb 3' of the nearest gene, eight were intragenic and four were in gene deserts. Whereas the AR occupied the same loci in C4-2B (castrate resistant) and LNCaP (androgen-dependent) PCa cells, differences between the two cell lines were observed in the response of nearby genes to androgens. Among the genes strongly stimulated by DHT in C4-2B cells – D-dopachrome tautomerase (DDT), Protein kinase C delta (PRKCD), Glutathione S- transferase theta 2 (GSTT2), Transient receptor potential cation channel subfamily V member 3 (TRPV3), and Pyrroline-5-carboxylate reductase 1 (PYCR1) – most were less strongly or hardly stimulated in LNCaP cells. Another AR target gene, ornithine aminotransferase (OAT), was AR-stimulated in a ligand-independent manner, since it was repressed by AR siRNA knockdown, but not stimulated by DHT. We also present evidence for in vivo as evidenced by their expression levels in prostate cancer tumors of various stages. Several AR target genes discovered in the present study, for example PRKCD and PYCR1, may open avenues in PCa research and aid the development of new approaches for disease management.AR genomic occupancy is similar between LNCaP and C4-2B cells and is not biased towards 5' gene flanking sequences. The AR transcriptionally regulates less than half the genes nearby AR-occupied regions, usually but not always, in a ligand-dependent manner. Most are stimulated and a few are repressed. In general, response is stronger in C4-2B compared to LNCaP cells. Some of the genes near AR-occupied regions appear to be regulated by the AR  Prostate Cancer (PCa) is the most commonly diagnosed non-cutaneous cancer and the second leading cause of cancer-related mortality in men . ProstatAlthough most recurrent PCa tumors are castrate-resistant, AR expression and function are maintained in advanced disease ,7 and thAs AR is a transcription factor, its oncogenic functions are likely mediated through specific target genes. Prostate specific antigen (PSA), the best studied AR target gene, is thought to contribute to PCa progression through its protease activity  and its AvaII in order to standardize all DNA fragments representing each AR-occupied region to one size. The AvaII fragments were amplified using ligation-mediated PCR with each of 36 possible nested primer combinations [To identify AR targets in PCa, we employed ChIP Display, a newly developed method for the identification of regions occupied by transcription factors in living cells . C4-2B hinations . The useinations . The PCRHaeIII digestion product from each of the two ChIPs was sequenced and mapped to human chromosome 17q25.3, 1.5-kb upstream of the MAFG gene and 2.5-kb 3' of the PYCR1 gene  were also occupied in LNCaP cells (a model of androgen-dependent PCa) Table , we suspAn emerging concept in PCa research is that ligand-independent AR-mediated gene expression contributes to the acquisition of a castrate-resistant growth state. If the derivation of C4-2B from LNCaP cells  were assin vitro CD-disclosed genes in 23 untreated primary PCa tumors, 17 primary tumors after 3 months of androgen ablation therapy and 7 AR-positive metastatic tumors. Expression of several of these genes was consistent with in vivo regulation by the AR. As shown in Figure in vitro  and not by five other KIAA1217 probesets present on the array  -28. WhilMany of the AR-occupied regions identified in the present study, which cannot be designated classical 5' promoter regions, were still close to annotated genes that they could potentially regulate. There is no consensus as to how far a transcription factor-binding region should be in order to be considered a putative cis-acting regulatory domain for a given gene. Values 1-kb, and up to 100-kb from the transcription start site have been used by various investigators ,30, but Comprehensive gene expression analysis is frequently employed for the discovery of target genes for transcription factors, including the AR ,34-36. SThe AR plays critical roles during all stages of PCa progression ,9,45. Itin vivo several of the ChIP Display-disclosed genes. This notion, however, remains tentative because the clinical material used for the microarray expression analysis is no longer available for confirmation by RT-PCR. To increase our confidence in the microarray data, we only considered results from probesets that are considered highly reliable (Affymetrix' grade A annotation). Results from the PCa biopsies were consistent with those from the in vitro gene expression analysis for the positively regulated genes PYCR1, DDT, PRKCD, and CRELD2. However, ALG12 and TRPV1, which appear to be stimulated by the AR in vivo, were not responsive to either DHT or AR siRNA in vitro. The in vivo and in vitro analyses were less well correlated for negatively regulated genes. Only one of six probesets interrogating KIAA1217 expression indicated repression in vivo, although this gene was strongly inhibited in vitro. CHRM1 was also strongly repressed by androgens in vitro, yet it was found to be stimulated in vivo. OAT, which appears to be downregulated by the AR in vivo, was stimulated in vitro in a ligand independent manner. It remains to be seen whether these inconsistencies result from differential requirements for AR-mediated gene stimulation/repression in vitro and in vivo, the presence of multiple splicing isoforms, or simply erroneous microarray expression scores. Be that as it may, further investigation of genes highlighted by our study, and especially genes for which results from the PCa biopsies are apparently inconsistent with the in vitro results, will have to start with validation of the regulation of such genes in PCa in vivo.In the present study, we included analysis of the ChIP Display-disclosed genes for their expression in PCa biopsies. This analysis provided evidence that AR regulates PSA (KLK3) remains the most well studied AR target gene in the PCa literature to date. Since its approval in 1986, serum PSA is routinely used to aid the early diagnosis and prognosis of PCa in men. However, AR-driven PSA expression alone does not fully explain the role of AR in PCa development and progression. Although additional AR target genes have been recently discovered, e.g., FKBP5  and TMPRin vitro data, we also demonstrate decreased PYCR1 expression in PCa biopsies from men undergoing androgen ablation therapy as compared to untreated controls. The highest PYCR1 expression in our PCa samples was found in biopsies from metastatic tumors, possibly as a result of atypical AR activation. Of the AR targets discovered in the present study, PYCR1 is a strong candidate for mediating the oncogenic action of AR signaling in PCa.Pyrroline-5-carboxylate reductase 1 (PYCR1) catalyzes the NAD(P)H-dependent conversion of pyrroline-5-carboxylate (P5C) to proline. Stimulation of PYCR1 by the AR could contribute to PCa progression because P5C is pro-apoptotic  and prolin vivo.Interestingly, another AR target gene discovered in this study also participates in proline metabolism. Ornithine aminotransferase (OAT) converts ornithine to glutamate γ-semialdehyde, which spontaneously cyclizes to form pyrroline-5-carboxylate (P5C), a proline precursor and the substrate for PYCR1. The functional evidence for AR-mediated regulation of OAT is weaker than that for PYCR1. While OAT mRNA was not significantly altered in response to DHT, it was repressed after siRNA-mediated knockdown of the AR, and also displayed a 3.3-fold higher basal expression in C4-2B as compared to LNCaP cells. Interestingly, OAT's expression pattern in our tumor samples does not suggest AR-mediated stimulation, but rather repression, possibly reflecting interactions of AR signaling with input from other cell types or components of the extracellular matrix, which only occur In this study, we mapped AR occupancy to a region 0.8-kb upstream of the gene encoding Protein Kinase C delta (PRKCD), which has received much attention in the PCa literature. PRKCD mRNA levels were higher in DHT-treated as compared to untreated PCa cell cultures and were reduced in PCa biopsies from patients undergoing androgen ablation therapy as compared to those from untreated patients. The observed high PRKCD mRNA levels in PCa metastases, which may reflect ligand-independent AR activation, possibly play a role in late stage disease because PRKCD is implicated in growth, migration and invasion of cancer cells, including PCa ,52. PRKCThe gene expression data form both the cell culture models and the clinical tumor samples suggest androgen-mediated stimulation of CRELD2 and DDT. Although a role for CRELD2 in carcinogenesis remains to be investigated, this Cysteine-rich with EGF-like Domains 2 (CRELD2) protein has been shown to interact with neuronal acetylcholine receptors . Likewisin vivo data does not support a role for MUC6 in PCa progression, MUC6 could still play a transient role during a short period of time not captured by our clinical samples.Among the few genes near AR-occupied regions that were repressed by DHT was MUC6. Mucin 6 is a secreted glycoprotein that forms a protective gel layer around the producing cell . Other min vitro data. These two genes are not only androgen stimulated but are also expressed in C4-2B cells more strongly than in LNCaP cells. TRPV3 is a member of the transient receptor potential (TRP) family of thermosensory ion channel genes. Another member of this family, TRPV6, potentiates calcium-dependent cell proliferation [Like the androgen-repressed MUC6, evidence for roles for the androgen-stimulated TRPV3 and GSTT2 genes in PCa progression is suggested only from our feration , and itsferation . Glutathferation .Two of the AR-occupied regions disclosed by ChIP Display were near loci previously linked to PCa: (i) the hereditary prostate cancer 1 (HPC1) locus, which has been mapped to 1q24-25 ; and  and their parental, androgen-dependent LNCaP cells, were obtained from ATCC. . The close relationship between the two cell lines was confirmed by microsatellite analysis at ten loci . The AvaII fragments were subjected to ligation-mediated PCR using each of 36 combinations of eight primers. Each primer had A or T at the +3 position of the AvaII site, and A,T,G, or C at the so-called +6 position, immediately internal to the AvaII site [HaeIII, HinfI and MspI (NEB), and sub-fragments were isolated by agarose gel electrophoresis and sequenced. The sequences were mapped to the human genome using the SSAHA program on ENSEMBL [We have recently described the CD procedure in detail . BrieflyaII site . In the  ENSEMBL .5 cells/well in 6 well plates) were cultured for two days in phenol red-free RPMI-1640 supplemented with 5% CSS. The cells were then transfected using OligofectAMINE (Invitrogen) with 100 nM of either AR-specific or non-specific siRNA . RNA quality was assessed spectrophotometrically and by agarose gel electrophoresis. High quality RNA 200–1000 ng) was reverse-transcribed with random hexamers using the Taqman reverse transcription reagents kit . cDNAs of interest were amplified using gene specific-primers  subfamily V member 3; PYCR1, pyrroline-5-carboxylate reductase 1; AP2A2, adaptor-related protein complex 2 alpha 2 subunit; ACBD6, Acyl-Coenzyme A binding domain containing 6; SIRT7, silent mating type information regulation 2 (sirtuin 7); MRFAP1, mof4 family associated protein 1; MAFG, v-maf musculoaponeurotic fibrosarcoma oncogene homolog G; OAT, ornithine amino transferase; CARKL, carbohydrate kinase like; MAN2B2, mannosidase 2 alpha B2; LHX4, LIM homeobox 4; ALG12, alpha 1,6 mannosyltransferase; CEP350, centrosomal protein 350 kDa; BAZ1B, bromodomain adjacent to zinc finger domain 1B; CLDN4, claudin-4; KIF1A, kinesin family member 1A; LHPP, phospholysine phosphohistidine inorganic pyrophosphate phosphatase; TRPV1, transient receptor potential (TRP) subfamily V member 1; WBSCR27, williams beuren syndrome chromosome region 27; SCL22A6, solute carrier family 22 (organic anion transporter); FZD9, frizzled homolog 9; QSCN6, quiescin Q6; MAP3K7IP1, mitogen-activated protein kinase kinase kinase 7 interacting protein 1; SYNGR1, synaptogyrin-1; MUC6, mucin 6; CHRM1, cholinergic receptor, muscarinic 1; WBSCR28, williams beuren syndrome chromosome region 28.The author(s) declare that they have no competing interests.UJ – Wrote the manuscript, contributed substantially to ChIP Display, gene expression analysis, and summary of the data.JP – Contributed substantially to ChIP Display, gene expression analysis and data interpretation.LJ – Generated the ChIP material for ChIP Display and participated in the experimental design.AB, SP – Processed the ChIP material for use in ChIP Display and participated in the experimental design.GB – Extracted and analyzed the gene expression data from the microarray experiment.JPC, AA – Contributed to ChIP Display.HIS, WLG, WDT – Designed and supervised the microarray studies on the clinical tumor samples.GAC, BF – Co-directed this study, corrected the manuscript.All authors read this manuscript.Oligonucleotides used in our studies. This file lists sequences of the oligonucleotides used for ChIP display, conventional ChIP assays, qPCR and siRNA.Click here for fileMicrosatellite analysis in LNCaP and C4-2B. This file provides the results of microsalletile analysis of C4-2B cells versus the parental LNCaP cells.Click here for fileComplete dataset and statistical analysis of DHT-responsive gene expression in C4-2B and LNCaP cells. This file provides the raw data, which is summarized in Figure Click here for fileComplete dataset and statistical analysis of basal gene expression in C4-2B versus LNCaP cells. This file provides the raw data, which is summarized in Figure Click here for file