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Abstract: G protein-coupled receptor 40 (GPR40) is a Gq-coupled receptor for free fatty acids predominantly expressed in pancreatic β-cells. In recent years, GPR40 agonists have been investigated for use as novel therapeutic agents in the treatment of type 2 diabetes. We discovered a novel small molecule GPR40 agonist, (3S)-3-ethoxy-3-(4-{[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]oxy}phenyl)propanoic acid (DS-1558). The GPR40-mediated effects of DS-1558 on glucose-stimulated insulin secretion were evaluated in isolated islets from GPR40 knock-out and wild-type (littermate) mice. The GPR40-mediated effects on glucose tolerance and insulin secretion were also confirmed by an oral glucose tolerance test in these mice. Furthermore, oral administration of DS-1558 (0.03, 0.1 and 0.3mg/kg) significantly and dose-dependently improved hyperglycemia and increased insulin secretion during the oral glucose tolerance test in Zucker fatty rats, the model of insulin resistance and glucose intolerance. Next, we examined the combination effects of DS-1558 with glucagon like peptide-1 (GLP-1). DS-1558 not only increased the glucose-stimulated insulin secretion by GLP-1 but also potentiated the maximum insulinogenic effects of GLP-1 after an intravenous glucose injection in normal Sprague Dawley rats. Furthermore, the glucose lowering effects of exendin-4, a GLP-1 receptor agonist, were markedly potentiated by the DS-1558 (3mg/kg) add-on in diabetic db/db mice during an intraperitoneal glucose tolerance test. In conclusion, our results indicate that add-on GPR40 agonists to GLP-1 related agents might be a potential treatment compared to single administration of these compounds. Therefore the combinations of these agents are a novel therapeutic option for type 2 diabetes. [Copyright &y& Elsevier]

Abstract: The ethoxyresorufin-O-deethylase (EROD) assay is a widely applied method for the evaluation of the dioxin-like activity of single substances and environmental samples. As for most enzyme assays, the specific activity is normally related to total protein contents, the determination of which has clear limitations in high-throughput assays. EROD induction potentials are usually expressed as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents, a substance highly toxic to humans. In order to compensate for these shortcomings, two modifications of the EROD protocol are proposed: (1) EROD activity is normalized to the metabolic activity of the cells as determined by a modified thiazolyl blue tetrazolium (MTT) assay and expressed as metabolic cell equivalents (MCE) based on MTT data rather than to protein contents. Via MCE data, cytotoxicity information can always be reported in parallel to EROD data; with the protocol presented here, MTT and EROD data are collected simultaneously. (2) Among several reference substances tested (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), β-naphthoflavone and benzo[a]pyrene), β-naphthoflavone proved to be the most suitable reference for the routine in vitro EROD assay, although TCDD has generally been preferred for purely scientific reasons. [Copyright &y& Elsevier]

We measured the relationship between CO2-induced seawater acidification, photo-physiological performance and intracellular pH (pHi) in a model cnidarian–dinoflagellate symbiosis – the sea anemone Aiptasia sp. – under ambient (289.94±12.54μatm), intermediate (687.40±25.10μatm) and high (1459.92±65.51μatm) CO2 conditions. These treatments represented current CO2 levels, in addition to CO2 stabilisation scenarios IV and VI provided by the Intergovernmental Panel on Climate Change (IPCC). Anemones were exposed to each treatment for two months and sampled at regular intervals. At each time-point we measured a series of physiological responses: maximum dark-adapted fluorescent yield of PSII (Fv/Fm), gross photosynthetic rate, respiration rate, symbiont population density, and light-adapted pHi of both the dinoflagellate symbiont and isolated host anemone cell. We observed increases in all but one photo-physiological parameter (Pgross:R ratio). At the cellular level, increases in light-adapted symbiont pHi were observed under both intermediate and high CO2 treatments, relative to control conditions (pHi 7.35 and 7.46 versus pHi 7.25, respectively). The response of light-adapted host pHi was more complex, however, with no change observed under the intermediate CO2 treatment, but a 0.3 pH-unit increase under the high CO2 treatment (pHi 7.19 and 7.48, respectively). This difference is likely a result of a disproportionate increase in photosynthesis relative to respiration at the higher CO2 concentration. Our results suggest that, rather than causing cellular acidosis, the addition of CO2 will enhance photosynthetic performance, enabling both the symbiont and host cell to withstand predicted ocean acidification scenarios. [ABSTRACT FROM AUTHOR]

Highlights: [•] Vicenin 2 is a potent inhibitor of α-glucosidase, PTP1B, and RLAR. [•] Vicenin 2 effectively inhibited the formation of advanced glycation end products. [•] Vicenin 2 suppressed glycation-induced protein oxidation. [•] Vicenin 2 also suppressed the formation of amyloid cross-β structures. [•] Vicenin 2 might be useful for the treatment of diabetes and its complications. [Copyright &y& Elsevier]

Abstract: Background: Plant lectins have attracted great interest in cancer studies due to their antitumor activities. These proteins or glycoproteins specifically and reversibly bind to different types of carbohydrates or glycoproteins. Breast cancer, which presents altered glycosylation of cell surface glycoproteins, is one of the most frequent malignant diseases in women. In this work, we describe the effect of the lectin Bauhinia forficata lectin (BfL), which was purified from B. forficata Link subsp. forficata seeds, on the MCF7 human breast cancer cellular line, investigating the mechanisms involved in its antiproliferative activity. Methods: MCF7 cells were treated with BfL. Viability and adhesion alterations were evaluated using flow cytometry and western blotting. Results: BfL inhibited the viability of the MCF7 cell line but was ineffective on MDA-MB-231 and MCF 10A cells. It inhibits MCF7 adhesion on laminin, collagen I and fibronectin, decreases α1, α6 and β1 integrin subunit expression, and increases α5 subunit expression. BfL triggers necrosis and secondary necrosis, with caspase-9 inhibition. It also causes deoxyribonucleic acid (DNA) fragmentation, which leads to cell cycle arrest in the G2/M phase and a decrease in the expression of the regulatory proteins pRb and p21. Conclusion: BfL shows selective cytotoxic effect and adhesion inhibition on MCF7 breast cancer cells. General significance: Cell death induction and inhibition of cell adhesion may contribute to understanding the action of lectins in breast cancer. [Copyright &y& Elsevier]

Abstract: Brain mitochondrial activity is centrally involved in the central control of energy balance. When studying mitochondrial functions in the brain, however, discrepant results might be obtained, depending on the experimental approaches. For instance, immunostaining experiments and biochemical isolation of organelles expose investigators to risks of false positive and/or false negative results. As an example, the functional presence of cannabinoid type 1 (CB1) receptors on brain mitochondrial membranes (mtCB1) was recently reported and rapidly challenged, claiming that the original observation was likely due to artifact results. Here, we addressed this issue by directly comparing the procedures used in the two studies. Our results show that the use of appropriate controls and quantifications allows detecting mtCB1 receptor with CB1 receptor antibodies, and that, if mitochondrial fractions are enriched and purified, CB1 receptor agonists reliably decrease respiration in brain mitochondria. These data further underline the importance of adapted experimental procedures to study brain mitochondrial functions. [Copyright &y& Elsevier]

Highlights: [•] Chloroquine synergizes sunitinib cytotoxicity in vitro and in vivo. [•] Chloroquine blocks sunitinib-induced autophagy. [•] Decreased survivin and increased caspase 3 activity partly explain this synergy. [•] Chloroquine enhances the anti-angiogenic effect of sunitinib. [•] Sunitinib–chloroquine combination increases nitric oxide and reduces ROS levels. [ABSTRACT FROM AUTHOR]

Abstract: Spiders of the Loxosceles genus represent a risk to human health due to the systemic and necrotic effects of their bites. The main symptoms of these bites vary from dermonecrosis, observed in the majority of cases, to occasional systemic hemolysis and coagulopathy. Although the systemic effects are well characterized, the mechanisms of cell death triggered by the venom of these spiders are poorly characterized. In this study, we investigated the cell death mechanisms induced by the whole venom of the spider Loxosceles similis in human skin fibroblasts. Our results show that the venom initiates an apoptotic process and a caspase cascade involving the initiator caspase-9 and the effector caspases-3, -6, and -7. [Copyright &y& Elsevier]

Highlights: [•] Mn exposure induces HtrA2 upregulation in rat striatum. [•] Upregulated HtrA2 was correlated with XIAP reduction and marked neuronal apoptosis. [•] HtrA2 inhibitor UCF-101 attenuated Mn-triggered XIAP downregulation. [•] UCF-101 partially protects neuronal cells from Mn-induced apoptosis. [ABSTRACT FROM AUTHOR]

Highlights: [•] Plasma treated titanium is better to cell viability than untreated titanium. [•] Cellular antioxidant response was more efficient on plasma treated titanium. [•] Plasma treatment could improve the titanium biocompatibility. [ABSTRACT FROM AUTHOR]

Highlights: [•] SMP-028 inhibits steroidogenesis in endocrine cells of rats in vitro. [•] SMP-028 C max,free are higher than IC50 values for steroidogenesis in vitro. [•] SMP-028 inhibition of steroidogenesis in vivo causes endocrine toxicology. [ABSTRACT FROM AUTHOR]

Abstract: To examine the role of intracellular labile iron pool (LIP), ferritin (Ft), and antioxidant defence in cellular resistance to oxidative stress on chronic adaptation, a new H2O2-resistant Jurkat T cell line “HJ16” was developed by gradual adaptation of parental “J16” cells to high concentrations of H2O2. Compared to J16 cells, HJ16 cells exhibited much higher resistance to H2O2-induced oxidative damage and necrotic cell death (up to 3mM) and had enhanced antioxidant defence in the form of significantly higher intracellular glutathione and mitochondrial ferritin (FtMt) levels as well as higher glutathione-peroxidase (GPx) activity. In contrast, the level of the Ft H-subunit (FtH) in the H2O2-adapted cell line was found to be 7-fold lower than in the parental J16 cell line. While H2O2 concentrations higher than 0.1mM fully depleted the glutathione content of J16 cells, in HJ16 cells the same treatments decreased the cellular glutathione content to only half of the original value. In HJ16 cells, H2O2 concentrations higher than 0.1mM increased the level of FtMt up to 4-fold of their control values but had no effect on the FtMt levels in J16 cells. Furthermore, while the basal cytosolic level of LIP was similar in both cell lines, H2O2 treatment substantially increased the cytosolic LIP levels in J16 but not in HJ16 cells. H2O2 treatment also substantially decreased the FtH levels in J16 cells (up to 70% of the control value). In contrast in HJ16 cells, FtH levels were not affected by H2O2 treatment. These results indicate that chronic adaptation of J16 cells to high concentrations of H2O2 has provoked a series of novel and specific cellular adaptive responses that contribute to higher resistance of HJ16 cells to oxidative damage and cell death. These include increased cellular antioxidant defence in the form of higher glutathione and FtMt levels, higher GPx activity, and lower FtH levels. Further adaptive responses include the significantly reduced cellular response to oxidant-mediated glutathione depletion, FtH modulation, and labile iron release and a significant increase in FtMt levels following H2O2 treatment. [Copyright &y& Elsevier]

Abstract: The aim of this study was to explore the anti-tumor potential of a polysaccharide isolated from Boschniakia rossica (BRP) in Hep2 human larynx squamous carcinoma cells. High performance size-exclusion chromatography analysis showed that BRP was a homogeneous polysaccharide and had a molecular weight of 22kDa. Total carbohydrate content in BRP was determined to be 96.9%, without the presence of protein and nucleic acid. BRP suppressed the proliferation of Hep2 cells in a time- and dose-dependent manner. Cell cycle analysis revealed that exposure to BRP (200μg/ml) caused a G0/G1 cell cycle arrest in Hep2 cells. Moreover, treatment with BRP at 100–400μg/ml for 24h induced a significant apoptosis Hep2 cells compared to untreated control cells, as determined by flow cytometry with annexin-V/propidium iodide double staining. Additionally, BRP treatment promoted the cleavage of pro-caspase-3, pro-caspase-8, and pro-caspase-9, coupled with increased expression of death receptor DR5 and Bax and reduced expression of Bcl-2. Taken together, our data demonstrate that BRP shows potent anti-tumor activity in human larynx squamous carcinoma, largely through induction of G0/G1 cell cycle arrest and apoptosis. Activation of both mitochondria-mediated and death receptor-mediated apoptosis pathways is involved in the cytotoxicity of BRP. [Copyright &y& Elsevier]

Highlights: [•] Carnosic acid (CA) lowered Aβ42/43 release in cultured human astrocytoma cells. [•] CA enhanced the expression of an α-secretase TACE without inducing other secretases. [•] Knockdown of TACE by siRNA partially recovered the CA-suppressed Aβ42/43 release. [•] CA may have a potential against Aβ-mediated diseases including Alzheimer's disease. [Copyright &y& Elsevier]

Highlights: [•] Yellow pigments monascin (MS) and ankaflavin (AK) are secondary metabolites derived from Monascus-fermented products. [•] MS and AK prevented fatty acid accumulation in oleic acid-induced steatosis in hepatocytes. [•] MS and AK attenuated high-fat diet-induced nonalcoholic fatty liver disease in mice. [•] MS and AK act as PPARα agonists to increase AMPK activity and beta-oxidation in hepatocytes. [Copyright &y& Elsevier]

Abstract: Modes and influencing factors of bovine serum albumin (BSA) and sulforaphane (SFN) interaction will help us understand the interaction mechanisms and functional changes of bioactive small molecule and biomacromolecule. This study investigated interaction mechanisms of BSA and SFN and associated antioxidant activity in three solvent systems of deionized water (dH2O), dimethyl sulfoxide (DMSO) and ethanol (EtOH), using Fourier transform infrared spectroscopy (FT-IR), fluorescence spectroscopy, synchronous fluorescence spectroscopy, DPPH and ABTS radical scavenging assays. The results revealed that SFN had ability to quench BSA's fluorescence in static modes, and to interact with BSA at both tyrosine (Tyr) and tryptophan (Trp) residues, while the Trp residues were highly sensitive, which was demonstrated by fluorescence at 340nm. Hydrophobic forces, hydrogen bonds and van der Waals interactions were all involved in BSA and SFN interaction, which were not significantly changed by three solvents. The binding constant values and binding site numbers were in a descending order of dH2O>DMSO>EtOH. The values of free energy change were in a descending order of dH2O>DMSO>EtOH, which indicated that the binding forces were in a descending order of dH2O>DMSO>EtOH. There was no significant difference in antioxidant activity between SFN and BSA–SFN. Moreover, three solvents had not significant influence on antioxidant activity of SFN and BSA–SFN. [Copyright &y& Elsevier]

Abstract: The Cancer/Testis Antigen (CTA), Prostate-associated Gene 4 (PAGE4), is a stress-response protein that is upregulated in prostate cancer (PCa) especially in precursor lesions that result from inflammatory stress. In cells under stress, translocation of PAGE4 to mitochondria increases while production of reactive oxygen species decreases. Furthermore, PAGE4 is also upregulated in human fetal prostate, underscoring its potential role in development. However, the proteins that interact with PAGE4 and the mechanisms underlying its pleiotropic functions in prostatic development and disease remain unknown. Here, we identified c-Jun as a PAGE4 interacting partner. We show that both PAGE4 and c-Jun are overexpressed in the human fetal prostate; and in cell-based assays, PAGE4 robustly potentiates c-Jun transactivation. Single-molecule Förster resonance energy transfer experiments indicate that upon binding to c-Jun, PAGE4 undergoes conformational changes. However, no interaction is observed in presence of BSA or unilamellar vesicles containing the mitochondrial inner membrane diphosphatidylglycerol lipid marker cardiolipin. Together, our data indicate that PAGE4 specifically interacts with c-Jun and that, conformational dynamics may account for its observed pleiotropic functions. To our knowledge, this is the first report demonstrating crosstalk between a CTA and a proto-oncogene. Disrupting PAGE4/c-Jun interactions using small molecules may represent a novel therapeutic strategy for PCa. [Copyright &y& Elsevier]

Highlights: [•] BPA for 24h promoted dendritic development of hippocampal neurons. [•] BPA for 24h increased the level of F-actin in dendrites of hippocampal neurons. [•] BPA promoted the expression of Rac1/Cdc42 but inhibited that of RhoA. [•] These BPA-induced effects were blocked by ERs antagonist and were suppressed by MEK1/2 inhibitor. [Copyright &y& Elsevier]

Highlights: [•] The difference between glucose–BSA and glycoaldehyde–BSA: effects on PC12 cells. [•] The protective effect mediated by GLP-1 receptor on AGE-induced neurotoxicity. [•] GLP-1 can reduce cell tau phosphorylation induced by high glucose or glucose–BSA. [•] GLP-1 regulated tau phosphorylation through a signaling pathway involving GSK-3β. [ABSTRACT FROM AUTHOR]

In order to evaluate the effects of meiotic stage on survival of vitrified goat oocytes, the Cryoloop method was used to cryopreserve goat immature germinal vesicle (GV) and meiosis II (MII) oocytes following in vitro maturation (IVM). The oocytes collected from local slaughter house were divided into two parts. In the first part, the immature cumulus oocyte complexes (COCs) were further divided into three groups: (1) untreated (control), (2) exposed to the vitrification and dilution solutions but without being plunged into liquid nitrogen (toxicity), or (3) vitrified by the Cryoloop method (vitrification). In the second part, the MII oocytes produced by IVM of immature COCs were also divided into three groups as the above immature COCs. Oocytes survival was assessed by morphological appearance, nuclear maturation, and developmental capability following parthenogenetic activation (PA). The experiment was repeated three times. Our data indicated the rates of oocytes with normal appearance and developmental capability in the toxicity or vitrification group were decreased as compared to the control oocytes. In the toxicity group, the rate of GV oocytes with normal morphology was 76.25%±3.37% and significantly less than that of MII oocytes (92.18%±1.94%, P <0.05). However, the cleavage rate of MII oocytes was not significantly different from that of GV oocytes in the toxicity group (68.23%±1.71% vs 67.59%±3.51%, P >0.05). Additionally, the percentage of GV oocytes developing to blastocyst was significantly less than that of MII oocytes in the toxicity group (13.84%±2.81% vs 29.78%±4.17%, P <0.05). The rate of vitrified/thawed GV oocytes with normal morphology was 60.37%±2.91% and significantly less than that of MII oocytes (82.91%±3.01%, P <0.05). Additionally, the cleavage rate of GV oocytes was also significantly less than that of MII oocytes in the vitrification group (42.81%±2.94% vs 57.91±1.06%, P <0.05). However, the blastocyst rate of vitrified/thawed GV oocytes was not significantly different from that of MII oocytes (8.51%±1.46% vs 12.41%±3.74%, P >0.05). In conclusion, although vitrification can greatly damage the structure and development capability of goat oocytes, the Cryoloop method can result in acceptable levels of survival and development of goat oocytes. Additionally, compared to immature GV goat oocytes, mature MII oocytes may be more tolerant to the vitrification process. [ABSTRACT FROM AUTHOR]

Abstract: Regulator of G-protein signaling (RGS) proteins potently suppress G-protein coupled receptor (GPCR) signal transduction by accelerating GTP hydrolysis on activated heterotrimeric G-protein α subunits. RGS4 is enriched in the CNS and is proposed as a therapeutic target for treatment of neuropathological states including epilepsy and Parkinson's disease. Therefore, identification of novel RGS4 inhibitors is of interest. An HEK293-FlpIn cell-line stably expressing M3-muscarinic receptor with doxycycline-regulated RGS4 expression was employed to identify compounds that inhibit RGS4-mediated suppression of M3-muscarinic receptor signaling. Over 300,000 compounds were screened for an ability to enhance Gαq-mediated calcium signaling in the presence of RGS4. Compounds that modulated the calcium response in a counter-screen in the absence of RGS4 were not pursued. Of the 1365 RGS4-dependent primary screen hits, thirteen compounds directly target the RGS-G-protein interaction in purified systems. All thirteen compounds lose activity against an RGS4 mutant lacking cysteines, indicating that covalent modification of free thiol groups on RGS4 is a common mechanism. Four compounds produce >85% inhibition of RGS4-G-protein binding at 100μM, yet are >50% reversible within a ten-minute time frame. The four reversible compounds significantly alter the thermal melting temperature of RGS4, but not G-protein, indicating that inhibition is occurring through interaction with the RGS protein. The HEK cell-line employed for this study provides a powerful tool for efficiently identifying RGS-specific modulators within the context of a GPCR signaling pathway. As a result, several new reversible, cell-active RGS4 inhibitors have been identified for use in future biological studies. [Copyright &y& Elsevier]

Abstract: The currently applied immunotherapy of type I allergy with aluminum hydroxide (alum) as adjuvant elicits – among other side effects – an initial IgE-boost. In contrast, CpG-oligodeoxynucleotides (ODNs) drive the immune response toward Th1. The biodegradable material protamine can spontaneously form nanoparticles together with such ODNs. Our aim was to investigate the immune response induced by protamine-based nanoparticles (proticles) with CpG-ODN as an allergen delivery system. Proticles complexed with Ara h 2 extracted from raw peanuts as model allergen were injected subcutaneously into naïve BALB/c mice. Ara h 2-specific antibodies were analyzed by ELISA and rat basophilic leukemia (RBL) cell assay. Cytokine levels were investigated in supernatants of stimulated splenocytes. The in vivo distribution after subcutaneous injection was examined via fluorescence imaging. BMDCs were stimulated with proticles, and expression of stimulation and maturation markers as well as cytokines in supernatants was investigated. A favorable increase in Ara h 2-specific IgG2a antibodies was found after immunization with proticles-Ara h 2, whereas Ara h 2-specific IgE was not detectable. Accordingly, the ratio of IL-5/IFN-gamma was low in this group. Granuloma formation was completely absent at injection sites of proticles. The distribution of Ara h 2 after subcutaneous injection was markedly decelerated when complexed to proticles. Stimulation of BMDCs with proticles-Ara h 2 caused upregulation of CD11c and CD80 as well as an increased IL-6 production. Our data suggest that biodegradable protamine-based nanoparticles with CpG-ODN counteract the Th2-dominated immune response induced by an allergen and therefore are suitable as novel carrier system for immunotherapy of allergy. [Copyright &y& Elsevier]

Abstract: Brans of 23 traditional and 12 improved (both red and white) rice varieties in Sri Lanka were screened for anti-amylase and anti-glycation activities in vitro. Varieties which showed the highest inhibitory activities at screening were further investigated for anti-glucosidase and glycation reversing as anti-diabetic properties. The same varieties were studied for selected antioxidant properties. Significantly high anti-amylase and anti-glycation activities were observed for bran extracts of red varieties compared to white varieties at screening. Traditional red rice varieties, Masuran, Sudu Heeneti, Dik Wee and Goda Heeneti, exhibited significant and dose dependent anti-amylase, anti-glycation and glycation reversing activities. These varieties also showed marked antioxidant properties. It is concluded that brans of Sri Lankan traditional red rice varieties Masuran, Sudu Heeneti, Dik Wee and Goda Heeneti may be potential food supplements for diabetes. [Copyright &y& Elsevier]

Abstract: In a mixed template, the presence of homologous target DNA sequences creates environments that almost inevitably give rise to artifacts and biases during PCR. Heteroduplexes, chimeras, and skewed template-to-product ratios are the exclusive attributes of mixed template PCR and never occur in a single template assay. Yet, multi-template PCR has been used without appropriate attention to quality control and assay validation, in spite of the fact that such practice diminishes the reliability of results. External and internal amplification controls became obligatory elements of good laboratory practice in different PCR assays. We propose the inclusion of an analogous approach as a quality control system for multi-template PCR applications. The amplification controls must take into account the characteristics of multi-template PCR and be able to effectively monitor particular assay performance. This study demonstrated the efficiency of a model mixed template as an adequate external amplification control for a particular PCR application. The conditions of multi-template PCR do not allow implementation of a classic internal control; therefore we developed a convenient semi-internal control as an acceptable alternative. In order to evaluate the effects of inhibitors, a model multi-template mix was amplified in a mixture with DNAse-treated sample. Semi-internal control allowed establishment of intervals for robust PCR performance for different samples, thus enabling correct comparison of the samples. The complexity of the external and semi-internal amplification controls must be comparable with the assumed complexity of the samples. We also emphasize that amplification controls should be applied in multi-template PCR regardless of the post-assay method used to analyze products. [Copyright &y& Elsevier]

Abstract: The androgens testosterone (T) and dihydrotestosterone (DHT), besides playing an important role in prostate development and growth, are also responsible for the development and progression of benign prostate hyperplasia (BPH) and prostate cancer. Therefore, the actions of these hormones can be antagonized by preventing the irreversible conversion of T into DHT by inhibiting 5α-reductase (5α-R). This has been a useful therapeutic approach for the referred diseases and can be achieved by using 5α-reductase inhibitors (RIs). Steroidal RIs, finasteride and dutasteride, are used in clinic for BPH treatment and were also proposed for chemoprevention of prostate cancer. Nevertheless, due to the increase in bone and muscle loss, impotency and occurrence of high-grade prostate tumours, it is important to seek for other potent and specific molecules with lower side effects. In the present work, we designed and synthesized steroids with the 3-keto-Δ4 moiety in the A-ring, as in the 5α-R substrate T, and with carboxamide, carboxyester or carboxylic acid functions at the C-17β position. The inhibitory 5α-R activity, in human prostate microsomes, as well as the anti-proliferative effects of the most potent compounds, in a human androgen-responsive prostate cancer cell line (LNCaP cells), were investigated. Our results showed that steroids 3, 4 and 5 are good RIs, which suggest that C-17β lipophylic amides favour 5α-R inhibition. Moreover, these steroids induce a decrease in cell viability of stimulated LNCaP cells, in a 5α-R dependent-manner, similarly to finasteride. [Copyright &y& Elsevier]

Abstract: Cholesteryl ester transfer protein (CETP), a key regulator of high-density lipoprotein (HDL) metabolism, induces HDL remodeling by transferring lipids between apolipoprotein B-containing lipoproteins and HDL, and/or by promoting lipid transfer between HDL subparticles. In this study, we investigated the mechanism as to how CETP induces the generation of lipid-poor particles (pre-β-HDL) from HDL, which increases ATP-binding cassette transporter 1-mediated cholesterol efflux. This CETP-dependent HDL remodeling is enhanced by the CETP modulator dalcetrapib both in plasma and isolated HDL. The interaction of dalcetrapib with cysteine 13 of CETP is required, since this effect was abolished when using mutant CETP in which cysteine 13 was substituted for a serine residue. Other thiol-containing compounds were identified as CETP modulators interacting with cysteine 13 of CETP. In order to mimic dalcetrapib-bound CETP, mutant CETP proteins were prepared by replacing cysteine 13 with the bulky amino acid tyrosine or tryptophan. The resultant mutants showed virtually no CETP-dependent lipid transfer activity but demonstrated preserved CETP-dependent pre-β-HDL generation. Overall, these data demonstrate that the two functions of CETP i.e., cholesteryl ester transfer and HDL remodeling can be uncoupled by interaction of thiol-containing compounds with cysteine 13 of CETP or by introducing large amino acid residues in place of cysteine 13. [Copyright &y& Elsevier]

Abstract: Resveratrol is a natural polyphenol that exerts potent effects to suppress atherosclerosis. However, its low concentration in plasma has placed this role in doubt. Thus, resveratrol effects might be dependent on its transport into vascular endothelium, a question not previously addressed in spite of its obvious and fundamental importance. Via high-performance liquid chromatography and liquid chromatography/mass spectrometry, we found that resveratrol was absorbed by human umbilical vein endothelial cells in a temperature-, concentration- and time-dependent manner, suggesting the involvement of passive diffusion and active transport. As determined by confocal laser scanning microscopy, resveratrol primarily distributed throughout the cytoplasm. Furthermore, resveratrol absorption was modulated by serum proteins and sodium-dependent glucose transporter 1 (SGLT1) yet inhibited by glucose (an SGLT1 substrate) and phlorizin (an SGLT1 selective inhibitor), as well as SGLT1 siRNA transfection. Additionally, Sprague–Dawley rats were intragastrically administrated with 100mg/kg of resveratrol and the concentration of resveratrol in blood vessels declined more slowly up to 24h compared to that in the blood. Our results suggested that resveratrol uptake by vascular endothelial cells involved both passive diffusion and an SGLT1-mediated process, at least partially. Moreover, the intracellular resveratrol pool may be more important than the serum level in vivo. These provide new insights into the cardiovascular benefits of resveratrol. [Copyright &y& Elsevier]

Highlights: [•] Isoproterenol (ISO) toxicity depends on muscle cell differentiation. [•] ISO triggers different toxicity pathways on differentiated H9c2 myoblasts. [•] Undifferentiated H9c2 cells challenged with ISO up-regulated SOD2 and Bcl-xL. [•] Differentiation of H9c2 is associated with differential regulation of stress responses, which regulates ISO effects. [Copyright &y& Elsevier]

Highlights: [•] Cyclometalated complexes of noble metals are promising anticancer prodrugs. [•] These compounds show improved stability and enhanced cytotoxicity. [•] Cyclometalated compounds are suitable for rational drug design. [•] Besides DNA, protein/enzyme interactions represent a major mode of action. [Copyright &y& Elsevier]

Abstract: We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α1-adrenoceptor (α1-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERK phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting β-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of α1A- and α1B-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization α1A-AR, but not α1B-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by β-arrestin 2 knockdown or concanavalin A treatment. α1D-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α1D antagonist BMY7378. Following activation by phenylephrine, α1D-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α1D-AR subtype was not observed via CypHer5 technology. The three α1-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α1A-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on β-arrestin 2 mediated internalization. [Copyright &y& Elsevier]

Abstract: Since the inflammatory response and oxidative stress are involved in the stroke cascade, we evaluated here the effects of Phycocyanobilin (PCB, the C-Phycocyanin linked tetrapyrrole) on PC12 cell survival, the gene expression and the oxidative status of hypoperfused rat brain. After the permanent bilateral common carotid arteries occlusion (BCCAo), the animals were treated with saline or PCB, taking samples 24h post-surgery. Global gene expression was analyzed with GeneChip Rat Gene ST 1.1 from Affymetrix; the expression of particular genes was assessed by the Fast SYBR Green RT-PCR Master Mix and Bioplex methods; and redox markers (MDA, PP, CAT, SOD) were evaluated spectrophotometrically. The PCB treatment prevented the H2O2 and glutamate induced PC12 cell injury assessed by the MTT assay, and modulated 190 genes (93 up- and 97 down-regulated) associated to several immunological and inflammatory processes in BCCAo rats. Furthermore, PCB positively modulated 19 genes mostly related to a detrimental pro-inflammatory environment and counteracted the oxidative imbalance in the treated BCCAo animals. Our results support the view of an effective influence of PCB on major inflammatory mediators in acute cerebral hypoperfusion. These results suggest that PCB has a potential to be a treatment for ischemic stroke for which further studies are needed. [Copyright &y& Elsevier]

Abstract: Background: Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are clinically used to counteract hyperglycemia. However, so far experienced unwanted side effects, such as weight gain, promote the search for new PPARγ activators. Methods: We used a combination of in silico, in vitro, cell-based and in vivo models to identify and validate natural products as promising leads for partial novel PPARγ agonists. Results: The natural product honokiol from the traditional Chinese herbal drug Magnolia bark was in silico predicted to bind into the PPARγ ligand binding pocket as dimer. Honokiol indeed directly bound to purified PPARγ ligand-binding domain (LBD) and acted as partial agonist in a PPARγ-mediated luciferase reporter assay. Honokiol was then directly compared to the clinically used full agonist pioglitazone with regard to stimulation of glucose uptake in adipocytes as well as adipogenic differentiation in 3T3-L1 pre-adipocytes and mouse embryonic fibroblasts. While honokiol stimulated basal glucose uptake to a similar extent as pioglitazone, it did not induce adipogenesis in contrast to pioglitazone. In diabetic KKAy mice oral application of honokiol prevented hyperglycemia and suppressed weight gain. Conclusion: We identified honokiol as a partial non-adipogenic PPARγ agonist in vitro which prevented hyperglycemia and weight gain in vivo. General significance: This observed activity profile suggests honokiol as promising new pharmaceutical lead or dietary supplement to combat metabolic disease, and provides a molecular explanation for the use of Magnolia in traditional medicine. [Copyright &y& Elsevier]

Abstract: Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27±3.9nM and 4.2±0.48nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported. [Copyright &y& Elsevier]

Abstract: The TNF-related apoptosis-inducing ligand (TRAIL or Apo2L) preferentially cause apoptosis of malignant cells in vitro and in vivo without severe toxicity. Therefore, TRAIL or agonist antibodies to the TRAIL DR4 and DR5 receptors are used in cancer therapy. However, many malignant cells are intrinsically resistant or acquire resistance to TRAIL. It has been previously proposed that the multidrug transporter P-glycoprotein (Pgp) might play a role in resistance of cells to intrinsic apoptotic pathways by interfering with components of ceramide metabolism or by modulating the electrochemical gradient across the plasma membrane. In this study we investigated whether Pgp also confers resistance toward extrinsic death ligands of the TNF family. To this end we focused our study on HeLa cells carrying a tetracycline-repressible plasmid system which shuts down Pgp expression in the presence of tetracycline. Our findings demonstrate that expression of Pgp is a significant factor conferring resistance to TRAIL administration, but not to other death ligands such as TNF-α and Fas ligand. Moreover, blocking Pgp transport activity sensitizes the malignant cells toward TRAIL. Therefore, Pgp transport function is required to confer resistance to TRAIL. Although the resistance to TRAIL-induced apoptosis is Pgp specific, TRAIL itself is not a direct substrate of Pgp. Pgp expression has no effect on the level of the TRAIL receptors DR4 and DR5. These findings might have clinical implications since the combination of TRAIL therapy with administration of Pgp modulators might sensitize TRAIL resistant tumors. [Copyright &y& Elsevier]

Abstract: Hepatocellular carcinoma (HCC) is one of the most aggressive malignant diseases and is highly resistant to conventional chemotherapy. Neferine, a major bisbenzylisoquinoline alkaloid derived from the embryos of Nelumbo nucifera, has been reported a few physiological activities. However, the mechanisms of anticancer effects are not well understood and its detailed activities on Hep3B cells have not been determined. Our results suggest that neferine exhibited cytotoxicity against HCC Hep3B cells, but not against HCC Sk-Hep1 and THLE-3, a normal human liver cell line. In addition, consistent with the induction of G1/S phase cell population in flow cytometry, downregulation of c-Myc, cyclin D1, D3, CDK4, E2F-1, as well as dephosphorlyation of cdc2 by western blot analysis, as evidenced by the appearance of cell cycle arrest, were observed in Hep3B cells treated with neferine. Our results demonstrated neferine induced ER stress and apoptosis, acting through multiple signaling cascades by the activation of Bim, Bid, Bax, Bak, Puma, caspases-3, -6, -7, -8 and PARP, and the protein expression levels of Bip, calnexin, PDI, calpain-2 and caspase-12 were also upregulated dramatically by neferine treatment. Overexpression of GFP-LC3B by neferine resulted in a diffuse cytosolic GFP fluorescence and the strong fluorescent spots, representing autophagosomes. The significant reduction of the migration in Hep3B cells and the capillary tube-like formation of HUVECs by neferine were also determined. These observations reveal that the therapeutic potential of neferine in treating HCC Hep3B cells, containing copies of hepatitis B virus (HBV) genomes. [Copyright &y& Elsevier]

Abstract: Background: Platelet activation in congestive heart failure (CHF) contributes to an increased risk for thromboembolic complications. Rivaroxaban, the first oral direct FXa inhibitor is approved in Europe for prevention and treatment of venous thrombosis, pulmonary embolism, and prevention of thromboembolic events in atrial fibrillation. As heart failure is an important risk factor for thromboembolism and increased platelet activation is common in heart failure, we investigated the potential effect of Rivaroxaban treatment on platelets in an experimental CHF model. Methods and results: Chronic myocardial infarction was induced in male Wistar rats by coronary ligation. Rats were randomized to placebo or Rivaroxaban (3 and 10mg/kg once daily). After 10 weeks platelet activation was assessed. Platelet-bound fibrinogen, detected by flow-cytometry, was significantly increased in CHF-Placebo (p <0.05) and reduced following treatment with Rivaroxaban (p <0.05 vs. CHF-Placebo). ADP-induced aggregation was significantly enhanced in CHF-Placebo vs. sham-operated animals (p <0.05) and normalized following chronic FXa inhibition (p <0.05 vs. CHF-Placebo). In separate in vitro experiments, attenuated platelet aggregation was present after incubating whole blood directly with Rivaroxaban but absent when the experiment was performed in platelet-rich plasma only. Thus, a direct effect on platelets could be excluded. Conclusion: Chronic direct factor Xa inhibition using Rivaroxaban reduces platelet activation in CHF rats by attenuating the secondary phase of ADP-induced platelet aggregation. Thus, Rivaroxaban may constitute a useful approach to prevent thromboembolic complications and reduce platelet activation in CHF at the same time. [Copyright &y& Elsevier]

Highlights: [•] Capsaicin triggers glutamate and dopamine release in the striatum of young rats only. [•] TRPV1R function is not enhanced under chemical and genetic ablation of CB1Rs. [•] Our data reveal a possible neurodevelopmental role for the TRPV1R in the rodent brain. [ABSTRACT FROM AUTHOR]

Highlights: [•] We analyzed the effect of VCA on proliferation and invasion in trophoblast. [•] Cell proliferations of trophoblast were increased at low concentration of VCA. [•] Low concentration of VCA increases MMP-2 expression in trophoblast. [•] The expressions of integrin subunits α5 and β1 were increased at 10pg/ml of VCA. [•] VCA promotes invasion ability of trophoblast via the Akt signaling. [Copyright &y& Elsevier]

Abstract: Proton efflux via voltage-gated proton channels (Hv1) is considered to mediate the charge compensation necessary to preserve NADPH oxidase activity during the respiratory burst. Using the Hv1 inhibitor Zn2+, we found that the PMA-induced respiratory burst of human neutrophils is inhibited when assessed as extracellular production of O2 − and H2O2, in accordance with literature studies, but, surprisingly, unaffected when measured as oxygen consumption or total (extracellular plus intracellular) H2O2 production. Furthermore, we show that inhibiting Hv1 with Zn2+ results in an increased production of intracellular ROS. Similar results, i.e. decreased extracellular and increased intracellular ROS production, were obtained using a human granulocyte-like cell line with severely impaired Hv1 expression. Acidic extracellular pH, which dampens proton efflux, also augmented intracellular production of H2O2. Zinc caused an increase in the rate but not in the extent of depolarization and cytosolic acidification indicating that mechanisms other than proton efflux take part in charge compensation. Our results suggest a hitherto unpredicted mechanism of charge compensation whereby, in the absence of proton efflux, part of O2 − generated within gp91phox in the plasma membrane is shunted intracellularly down electrochemical gradient to dampen excessive depolarization. This would preserve NADPH oxidase activity under conditions such as the inflammatory exudate in which the acidic pH hinders charge compensation by proton efflux. [Copyright &y& Elsevier]

Abstract: The development of tumor-selective drugs with low systemic toxicity has always been a major challenge in cancer treatment. Our group previously identified the 7,8-dihydroxy-4-methylcoumarin (DHMC) as a potential chemotherapeutic agent due to its potent, selective anti-proliferative and apoptosis-inducing effects on several cancer cell lines over peripheral blood mononuclear cells. However, there are still no published reports that can explain such selectivity of action. Herein, we addressed this question by using the U-937 promonocytic leukemia cell line, which can be forced to differentiate into a monocyte-like phenotype in vitro. U-937 cells differentiation is dependent on the nuclear expression of p21Cip1/WAF1, a protein that is absent in immature U-937 cells but present in both the nucleus and the cytoplasm of normal DHMC-resistant monocytes. Considering that induction of differentiation rendered U-937 cells resistant to DHMC, we evaluated the possible causal role of cytoplasmic p21Cip1/WAF1 in the onset of such resistance by employing U-937 cells stably transfected with a ZnCl2-inducible p21Cip1/WAF1 variant lacking the nuclear localization signal (U-937/CB6-ΔNLS-p21 cells). Expression of cytoplasmic p21Cip1/WAF1 did not induce differentiation of the cells but turned them resistant to DHMC through inhibition of JNK, a crucial mediator of DHMC-induced apoptosis in U-937 cells. Sub-acute toxicity evaluation of DHMC in Balb/c mice indicated that DHMC administered intraperitoneally at doses up to 100mg/kg induced no systemic damage. Collectively, our results explain for the first time the selective cytotoxicity of DHMC for tumor cells over normal monocytes, and encourage further in vivo studies on this compound as potential anti-leukemic agent. [Copyright &y& Elsevier]

Highlights: [•] Leptin and ghrelin protect hippocampal neurons from Aβ oligomers-induced toxicity. [•] Leptin and ghrelin prevent oxidative stress and mitochondrial dysfunction. [•] Leptin and ghrelin revert GSK3β activation induced by Aβ oligomers. [•] Neuroprotection by leptin and ghrelin occurs in a receptor-mediated manner. [ABSTRACT FROM AUTHOR]

Highlights: [•] Inhibiting HIV-1 integrase (IN) DNA binding is a way to develop new anti-HIV drugs. [•] The first high-throughput assay is developed to screen IN DNA binding inhibitors. [•] The economical assay shows good agreements with results obtained by other assays. [•] A new IN binding inhibitor nigranoic acid was identified using this assay. [•] Provide a proof-of-concept of using the strategy to develop new drugs against HIV. [Copyright &y& Elsevier]

Abstract: The histone acetyltransferase Rtt109 is the sole enzyme responsible for acetylation of histone H3 lysine 56 (H3K56) in fungal organisms. Loss of Rtt109 renders fungal cells extremely sensitive to genotoxic agents, and prevents pathogenesis in several clinically important species. Here, via a high throughput chemical screen of >300,000 compounds, we discovered a chemical inhibitor of Rtt109 that does not inhibit other acetyltransferase enzymes. This compound inhibits Rtt109 regardless of which histone chaperone cofactor protein (Asf1 or Vps75) is present, and appears to inhibit Rtt109 via a tight-binding, uncompetitive mechanism. [Copyright &y& Elsevier]

Abstract: Cell death in response to oxidative stress plays a role in a variety of neurodegenerative diseases and can be studied in detail in the neuronal cell line HT22, where extracellular glutamate causes glutathione depletion by inhibition of the glutamate/cystine antiporter system xc −, elevation of reactive oxygen species and eventually programmed cell death caused by cytotoxic calcium influx. Using this paradigm, we screened 54 putative extracellular peptide or small molecule ligands for effects on cell death and identified extracellular cyclic guanosine monophosphate (cGMP) as a protective substance. Extracellular cGMP was protective, whereas the cell-permeable cGMP analog 8-pCPT-cGMP or the inhibition of cGMP degradation by phosphodiesterases was toxic. Interestingly, metabolites GMP and guanosine were even more protective than cGMP and the inhibition of the conversion of GMP to guanosine attenuated its effect, suggesting that GMP offers protection through its conversion to guanosine. Guanosine increased system xc − activity and cellular glutathione levels in the presence of glutamate, which can be explained by transcriptional upregulation of xCT, the functional subunit of system xc −. However, guanosine also provided protection when added late in the cell death cascade and significantly reduced the number of calcium peaking cells, which was most likely not mediated by transcriptional mechanisms. We observed no changes in the classical protective pathways such as phosphorylation of Akt, ERK1/2 or induction of Nrf2 or ATF4. We conclude that extracellular guanosine protects against endogenous oxidative stress by two probably independent mechanisms involving system xc − induction and inhibition of cytotoxic calcium influx. [Copyright &y& Elsevier]

Abstract: Protein oxidation, irreversible modification, and inactivation may play key roles in various neurodegenerative disorders. Therefore, we studied the effects of the potentially in vivo occurring nitric oxide-related species on two different markers of protein oxidation: protein carbonyl generation on bovine serum albumine (BSA) and loss of activity of a cysteine-dependent protease, papain, in vitro by using Angeli''s salt, papanonoate, SIN-1, and S-nitrosoglutathione (GSNO) as donors of nitroxyl, nitric oxide, peroxynitrite, and nitrosonium ions, respectively. Angeli''s salt, SIN-1, and papanonoate (0–1000 μM) all generated a concentration-dependent increase in carbonyl formation on BSA (107, 60, and 45%, respectively). GSNO did not affect carbonyl formation. Papain was inhibited by Angeli''s salt, SIN-1, papanonoate, and GSNO with IC50 values of 0.62, 2.3, 54, and 80 μM, respectively. Angeli''s salt (3.16 μM)-induced papain inactivation was only partially reversible, while the effects of GSNO (316 μM) and papanonoate (316 μM) were reversible upon addition of excess DTT. The Angeli''s salt-mediated DTT-irreversible inhibition of papain was prevented by GSNO or papanonoate pretreatment, hypothetically through mixed disulfide formation or S-nitrosylation of the catalytically critical thiol group of papain. These results, for the first time, compare the generation of carbonyls in proteins by Angeli''s salt, papanonoate, and SIN-1. Furthermore, these results suggest that S-nitrosothiols may have a novel function in protecting critical thiols from irreversible oxidative damage. [Copyright &y& Elsevier]

Abstract: It has been well documented that the activation of c-Jun N-terminal protein kinase (JNK) pathway and caspase-3 signal are involved in the delayed neuronal cell death in cerebral ischemia. In this study, we first detected the activation pattern of JNK signaling including mixed lineage kinase (MLK)3, mitogen-activated protein kinase kinase (MKK)7 and JNK3 in hippocampal CA1 and CA3/DG regions at various time points after 15 min of ischemia. These results indicated that cerebral ischemia induced the continuous activation of MLK3/MKK7/JNK3 cascade, which all had two active waves only in the CA1 region. We also detected the phosphorylation of JNK substrates c-Jun and Bcl-2, and the activation of a key protease of caspase-3 in CA1 region, which only had one active peak, respectively. Because K252a has recently been shown to be a potent inhibitor of MLK3 activity both in vivo and in vitro, we further examined the possible effects and mechanism of this interesting drug in cerebral ischemia. In our present paper, we found that administration of K252a 20 min prior to ischemia inhibited MLK3/MKK7/JNK3 signaling, Bcl-2 phosphorylation, the activation of c-Jun and caspase-3, but had no significant effects on these protein expressions. Additionally, pretreatment of K252a significantly increased the number of the surviving CA1 pyramidal cells at 5 days of reperfusion. Our results suggest that K252a play a neuroprotective role in ischemic injury via inhibition of the JNK pathway, involving the death effector of caspase-3. Thus, JNK signaling may eventually emerge as a prime target for novel therapeutic approaches to treatment of ischemic stroke, and K252a may serve as a potential and important neuroprotectant in therapeutic aspect in ischemic stroke. [Copyright &y& Elsevier]

While the comet assay is used to detect DNA damage in isolated cells following exposure to chemicals in vitro, few publications report the use of the procedure in liver cells isolated from mice. Our initial efforts to use the assay to assess DNA damage in mouse hepatocytes maintained on collagen-coated dishes were hampered by high levels of baseline damage in controls, which appeared to result from mechanical damage sustained during the dislodgement of adherent cells in the early stages of the assay protocol. Here we describe an efficient version of the comet assay in cultured mouse hepatocytes that involves careful recovery of cells using a “scraping” buffer supplemented with 10% high purity grade DMSO. Use of this buffer strongly diminished the frequency of false positives. Using the industrial reagent styrene as a positive control in the optimised procedure, non-cytotoxic concentrations of this substance (2.5–10 mM) significantly increased mean comet tail length, area, and moment. Co-incubation with the CYP inhibitor SKF-525A strongly attenuated these effects of styrene. Collectively, these findings confirm this method is highly suitable for the detection of DNA damage by bioactivation-dependent compounds in freshly isolated mouse hepatocytes. [Copyright &y& Elsevier]

UDP-N-acetylmuramyl-l-alanine ligase (MurC) is an essential bacterial enzyme involved in peptidoglycan biosynthesis and a target for the discovery of novel antibacterial agents. As a result of a high-throughput screen (HTS) against a chemical library for inhibitors of MurC, a series of benzofuran acyl-sulfonamides was identified as potential leads. One of these compounds, Compound A, inhibited Escherichia coli MurC with an IC50 of 2.3 μM. Compound A exhibited time-dependent, partially reversible inhibition of E. coli MurC. Kinetic studies revealed a mode of inhibition consistent with the compound acting competitively with the MurC substrates ATP and UDP-N-acetyl-muramic acid (UNAM) with a Ki of 4.5 μM against ATP and 6.3 μM against UNAM. Fluorescence binding experiments yielded a Kd of 3.1 μM for the compound binding to MurC. Compound A also exhibited high-affinity binding to bovine serum albumin (BSA) as evidenced by a severe reduction in MurC inhibition upon addition of BSA. This finding is consistent with the high lipophilicity of the compound. Advancement of this compound series for further drug development will require reduction of albumin binding. [Copyright &y& Elsevier]

2-Arachidonoylglycerol (2-AG) is an endogenous cannabinoid that binds to CB1 and CB2 cannabinoid receptors, inducing cannabimimetic effects. However, the cannabimimetic effects of 2-AG are weak in vivo due to its rapid enzymatic hydrolysis. The enzymatic hydrolysis of 2-AG has been proposed to mainly occur by monoglyceride lipase (monoacylglycerol lipase). Fatty acid amide hydrolase (FAAH), the enzyme responsible for the hydrolysis of N-arachidonoylethanolamide (AEA), is also able to hydrolyse 2-AG. In the present study, we investigated the hydrolysis of endocannabinoids in rat cerebellar membranes and observed that enzymatic activity towards 2-AG was 50-fold higher than that towards AEA. Furthermore, various inhibitors for 2-AG hydrolase activity were studied in rat cerebellar membranes. 2-AG hydrolysis was inhibited by methyl arachidonylfluorophosphonate, hexadecylsulphonyl fluoride and phenylmethylsulphonyl fluoride with ic50 values of 2.2 nM, 241 nM and 155 μM, respectively. Potent FAAH inhibitors, such as OL-53 and URB597, did not inhibit the hydrolysis of 2-AG, suggesting that 2-AG is inactivated in rat cerebellar membranes by an enzyme distinct of FAAH. The observation that the hydrolysis of 1(3)-AG and 2-AG occurred at equal rates supports the role of MGL in 2-AG inactivation. This enzyme assay provides a useful method for future inhibition studies of 2-AG degrading enzyme(s) in brain membrane preparation having considerably higher MGL-like activity when compared to FAAH activity. [Copyright &y& Elsevier]

Cannabinoids have been shown to impair cognition in vivo and block long-term potentiation (LTP), a candidate experimental model of learning and memory in vitro, via cannabinoid receptor (CB1) activation. cis-Oleamide (cOA) is an endogenous sleep-inducing lipid with putative cannabinomimetic properties. We hypothesise that cOA is cannabinomimetic and perform a comparative study with synthetic and endogenous cannabinoids on their effects on synaptic conditioning via two different patterns of stimulation in the hippocampal slice. CB1 agonists, R(+)-WIN55212-2 and anandamide, but not cOA blocked high frequency stimulation (HFS)-LTP. R(+)-WIN55212-2 and cOA (stereoselectively) attenuated responses to θ-burst-LTP, while anandamide did not. The anandamide transport inhibitor, AM404, attenuated HFS-LTP, an effect reversed by the CB1 receptor antagonist SR141716A but not mimicked by the vanilloid receptor agonist capsaicin. TFNO, an inhibitor of fatty acid amide hydrolase (FAAH), the enzyme responsible for degrading anandamide, failed to block HFS-LTP alone or in combination with cOA. On the contrary, this combination was as effective as cOA on its own in attenuating θ-burst-LTP. cOA effects on θ-burst-LTP were prevented in the presence of the GABAA receptor blocker picrotoxin, but not by pretreatment with SR141716A. These findings suggest that cOA neither directly activates CB1 receptors nor acts via the proposed “entourage” effect [Nature 389 (1997) 25] to increase titres of anandamide through FAAH inhibition. The selective effects of cOA on θ-burst-conditioning may reflect modulation of GABAergic transmission. Anandamide uptake inhibition, but not blockade of FAAH, effectively increases synaptic concentrations of endocannabinoids. [Copyright &y& Elsevier]