Impact of Basolateral Multidrug Resistance-Associated Protein (Mrp) 3 and Mrp4 on the Hepatobiliary Disposition of Fexofenadine in Perfused Mouse Livers

The disposition of fexofenadine, a commonly-used antihistamine drug, is governed primarily by active transport. Biliary excretion of the parent compound is the major route of systemic clearance. Previous studies demonstrated that fexofenadine hepatic uptake is mediated by organic anion transporting polypeptides. Recently, we showed that in mice fexofenadine is excreted into bile primarily by Mrp2 (Abcc2). In the present study, the role of Mrp3 (Abcc3) and Mrp4 (Abcc4) in the hepatobiliary disposition of fexofenadine was examined in knockout mice using in situ liver perfusion. Compared to wild-type mice, basolateral excretion of fexofenadine was impaired resulting in a ~50% decrease in perfusate recovery in Abcc3 ( − / − ) mice; in contrast, fexofenadine hepatobiliary disposition was unaltered in Abcc4 ( − / − ) mice. As expected, in Abcc2 ( − / − ) mice, fexofenadine was redirected from the canalicular to the basolateral membrane for excretion. In Abcc2 ( − / − )/Abcc3 ( − / − ) double knock-out mice, fexofenadine biliary excretion was impaired, but perfusate recovery was similar to wild-type mice, and more than 2-fold higher than in Abcc3 ( − / − ) mice, presumably due to compensatory basolateral transport mechanism(s). These results demonstrate that multiple transport proteins are involved in the hepatobiliary disposition of fexofenadine. In addition to Mrp2 and Mrp3, other transport proteins play an important role in the biliary and hepatic basolateral excretion of this zwitterionic drug

Crystal Structures of ABL-Related Gene (ABL2) in Complex with Imatinib, Tozasertib (VX-680), and a Type I Inhibitor of the Triazole Carbothioamide Class†

ABL2 (also known as ARG (ABL related gene)) is closely related to the well-studied Abelson kinase cABL. ABL2 is involved in human neoplastic diseases and is deregulated in solid tumors. Oncogenic gene translocations occur in acute leukemia. So far no structural information for ABL2 has been reported. To elucidate structural determinants for inhibitor interaction, we determined the cocrystal structure of ABL2 with the oncology drug imatinib. Interestingly, imatinib not only interacted with the ATP binding site of the inactive kinase but was also bound to the regulatory myristate binding site. This structure may therefore serve as a tool for the development of allosteric ABL inhibitors. In addition, we determined the structures of ABL2 in complex with VX-680 and with an ATP-mimetic type I inhibitor, which revealed an interesting position of the DFG motif intermediate between active and inactive conformations, that may also serve as a template for future inhibitor design

Combined effects of GSTP1 and MRP1 in melanoma drug resistance

Glutathione-S-transferase Pi1 (GSTP1) and multidrug resistance protein 1 (MRP1) are overexpressed in melanoma, a skin cancer notoriously resistant to all current modalities of cancer therapy. To investigate the involvement of these detoxifying enzymes in the drug resistance of melanoma, an inducible (Tet-On™ system) antisense (AS) RNA strategy was used to specifically inhibit GSTP1 expression in A375 cells, a human melanoma cell line expressing high levels of GSTP1 and MRP1. Stable transfectant clones were established and analysed for GSTP1 inhibition by AS RNA. The clone A375-ASPi1, presenting a specific 40% inhibition of GSTP1 expression in the presence of doxycycline, was selected. Lowering the GSTP1 level significantly increased (about 3.3-fold) the sensitivity of A375-ASPi1 cells to etoposide. Inhibitors of glutathione synthesis (BSO), GSTs (curcumin, ethacrynic acid), and also of MRPs (MK571, sulphinpyrazone) improved the sensitising effect of GSTP1 AS RNA. All these inhibitors had stronger sensitising effects in control cells expressing high GSTP1 level (A375-ASPi1 cells in the absence of doxycycline). In conclusion, GSTP1 can act in a combined fashion with MRP1 to protect melanoma cells from toxic effects of etoposide

p-Glycoprotein ABCB5 and YB-1 expression plays a role in increased heterogeneity of breast cancer cells: correlations with cell fusion and doxorubicin resistance

<p>Abstract</p> <p>Background</p> <p>Cancer cells recurrently develop into acquired resistance to the administered drugs. The iatrogenic mechanisms of induced chemotherapy-resistance remain elusive and the degree of drug resistance did not exclusively correlate with reductions of drug accumulation, suggesting that drug resistance may involve additional mechanisms. Our aim is to define the potential targets, that makes drug-sensitive MCF-7 breast cancer cells turn to drug-resistant, for the anti-cancer drug development against drug resistant breast cancer cells.</p> <p>Methods</p> <p>Doxorubicin resistant human breast MCF-7 clones were generated. The doxorubicin-induced cell fusion events were examined. Heterokaryons were identified and sorted by FACS. In the development of doxorubicin resistance, cell-fusion associated genes, from the previous results of microarray, were verified using dot blot array and quantitative RT-PCR. The doxorubicin-induced expression patterns of pro-survival and pro-apoptotic genes were validated.</p> <p>Results</p> <p>YB-1 and ABCB5 were up regulated in the doxorubicin treated MCF-7 cells that resulted in certain degree of genomic instability that accompanied by the drug resistance phenotype. Cell fusion increased diversity within the cell population and doxorubicin resistant MCF-7 cells emerged probably through clonal selection. Most of the drug resistant hybrid cells were anchorage independent. But some of the anchorage dependent MCF-7 cells exhibited several unique morphological appearances suggesting minor population of the fused cells maybe de-differentiated and have progenitor cell like characteristics.</p> <p>Conclusion</p> <p>Our work provides valuable insight into the drug induced cell fusion event and outcome, and suggests YB-1, GST, ABCB5 and ERK3 could be potential targets for the anti-cancer drug development against drug resistant breast cancer cells. Especially, the ERK-3 serine/threonine kinase is specifically up-regulated in the resistant cells and known to be susceptible to synthetic antagonists.</p

Psychopathic traits and offender characteristics – a nationwide consecutive sample of homicidal male adolescents

<p>Abstract</p> <p>Background</p> <p>The aim of the study was to evaluate psychopathy-like personality traits in a nationwide consecutive sample of adolescent male homicide offenders and to compare the findings with those of a randomly sampled adult male homicide offender group. A further aim was to investigate associations between psychopathic traits and offender and offence characteristics in adolescent homicides.</p> <p>Methods</p> <p>Forensic psychiatric examination reports and crime reports of all 15 to19- year- old male Finnish offenders who had been subjected to a forensic psychiatric examination and convicted for a homicide during 1995–2004 were collected (n = 57). A random sample of 57 adult male homicide offenders was selected as a comparison group. Offence and offender characteristics were collected from the files and a file-based assessment of psychopathic traits was performed using the Hare Psychopathy Checklist-Revised (PCL-R) by trained raters.</p> <p>Results</p> <p>No significant differences existed between the adolescents and adults in PCL-R total scores, factor 2 (social deviance) scores, or in facets 3 (lifestyle) and 4 (antisocial). Adults scored significantly higher on factor 1 (interpersonal/affective) and facets 1 (interpersonal) and 2 (affective). The adolescent group was divided into two subgroups according to PCL-R total scores. One in five homicidal male adolescents met criteria for psychopathic personality using a PCL-R total score of 26 or higher. These boys significantly more often had a crime history before the index homicide, more frequently used excessive violence during the index homicide, more rarely lived with both parents until 16 years of age, had more institutional or foster home placements in childhood, had more school difficulties, more often had received special education, and, more often had contact with mental health services prior to age 18 years than boys scoring low on the PCL-R. They also more often had parental criminal history as well as homicide history of parents or near relatives than the group scoring low on the PCL-R.</p> <p>Conclusion</p> <p>Homicidal boys behaved as antisocially as the homicidal adults. The adults, however, showed more both affective and interpersonal features of psychopathy. Homicidal adolescents with psychopathy-like personality character form a special subgroup among other homicidal youngsters. Recognizing their characteristics, especially in life course development, would facilitate effective prevention and intervention efforts.</p

Proteins with Complex Architecture as Potential Targets for Drug Design: A Case Study of Mycobacterium tuberculosis

Lengthy co-evolution of Homo sapiens and Mycobacterium tuberculosis, the main causative agent of tuberculosis, resulted in a dramatically successful pathogen species that presents considerable challenge for modern medicine. The continuous and ever increasing appearance of multi-drug resistant mycobacteria necessitates the identification of novel drug targets and drugs with new mechanisms of action. However, further insights are needed to establish automated protocols for target selection based on the available complete genome sequences. In the present study, we perform complete proteome level comparisons between M. tuberculosis, mycobacteria, other prokaryotes and available eukaryotes based on protein domains, local sequence similarities and protein disorder. We show that the enrichment of certain domains in the genome can indicate an important function specific to M. tuberculosis. We identified two families, termed pkn and PE/PPE that stand out in this respect. The common property of these two protein families is a complex domain organization that combines species-specific regions, commonly occurring domains and disordered segments. Besides highlighting promising novel drug target candidates in M. tuberculosis, the presented analysis can also be viewed as a general protocol to identify proteins involved in species-specific functions in a given organism. We conclude that target selection protocols should be extended to include proteins with complex domain architectures instead of focusing on sequentially unique and essential proteins only

Low Concentration of Sodium Butyrate from Ultrabraid+NaBu suture, Promotes Angiogenesis and Tissue Remodelling in Tendon-bones Injury

Sodium butyrate (NaBu), a form of short-chain fatty acid (SCFA), acts classically as a potent anti-angiogenic agent in tumour angiogenesis models, some authors demonstrated that low concentrations of NaBu may contribute to healing of tendon-bone injury in part at least through promotion of tissue remodelling. Here, we investigated the effects of low-range concentrations of NaBu using in vitro and in vivo assays using angiogenesis as the primary outcome measure and the mechanisms through which it acts. We demonstrated that NaBu, alone or perfused from the UltraBraid+NaBu suture was pro-angiogenic at very low-range doses promoting migration, tube formation and cell invasion in bovine aortic endothelial cells (BAECs). Furthermore, cell exposure to low NaBu concentrations increased expression of proteins involved in angiogenic cell signalling, including p-PKCβ1, p-FAK, p-ERK1/2, p-NFκβ, p-PLCγ1 and p-VEGFR2. In addition, inhibitors of both VEGFR2 and PKCβ1 blocked the angiogenic response. In in vivo assays, low concentrations of NaBu induced neovascularization in sponge implants in mice, evidenced by increased numbers of vessels and haemoglobin content in these implants. The findings in this study indicate that low concentrations of NaBu could be an important compound to stimulate angiogenesis at a site where vasculature is deficient and healing is compromised

Characterizing the Syphilis-Causing Treponema pallidum ssp. pallidum Proteome Using Complementary Mass Spectrometry

YesBackground. The spirochete bacterium Treponema pallidum ssp. pallidum is the etiological agent of syphilis, a chronic multistage disease. Little is known about the global T. pallidum proteome, therefore mass spectrometry studies are needed to bring insights into pathogenicity and protein expression profiles during infection. Methodology/Principal Findings. To better understand the T. pallidum proteome profile during infection, we studied T. pallidum ssp. pallidum DAL-1 strain bacteria isolated from rabbits using complementary mass spectrometry techniques, including multidimensional peptide separation and protein identification via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) and electrospray ionization (ESI-LTQ-Orbitrap) tandem mass spectrometry. A total of 6033 peptides were detected, corresponding to 557 unique T. pallidum proteins at a high level of confidence, representing 54% of the predicted proteome. A previous gel-based T. pallidum MS proteome study detected 58 of these proteins. One hundred fourteen of the detected proteins were previously annotated as hypothetical or uncharacterized proteins; this is the first account of 106 of these proteins at the protein level. Detected proteins were characterized according to their predicted biological function and localization; half were allocated into a wide range of functional categories. Proteins annotated as potential membrane proteins and proteins with unclear functional annotations were subjected to an additional bioinformatics pipeline analysis to facilitate further characterization. A total of 116 potential membrane proteins were identified, of which 16 have evidence supporting outer membrane localization. We found 8/12 proteins related to the paralogous tpr gene family: TprB, TprC/D, TprE, TprG, TprH, TprI and TprJ. Protein abundance was semi-quantified using label-free spectral counting methods. A low correlation (r = 0.26) was found between previous microarray signal data and protein abundance. Conclusions. This is the most comprehensive description of the global T. pallidum proteome to date. These data provide valuable insights into in vivo T. pallidum protein expression, paving the way for improved understanding of the pathogenicity of this enigmatic organism.This work was supported by the grants from the Flanders Research Foundation, SOFI-B Grant to CRK, http://www.fwo.be/, a Public Health Service Grant from the National Institutes of Health to CEC, (grant # AI-051334), https://www.nih.gov/ and a grant from the Grant Agency of the Czech Republic to DS and MS (P302/12/0574, GP14-29596P), https:// gacr.cz/