The structures of garcinones a,b and c: Three new xanthones from Garcinia mangostana

Three new tetraoxygenated xanthones (garcinones A, B and C), each disubstituted with C₅-units, have been isolated from the chloroform extract of the fruit-hulls of Garcinia mangostana. Their structures were established by a combination of spectral interpretation and chemical correlation.     

Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung

A radioligand binding assay has been established to study leukotriene specific binding sites in the guinea pig and rabbit tissues. Using high specific activity [³H]-leukotriene D₄ ([³H]-LTD₄), in the presence or absence of unlabeled LTD₄, the diastereoisomer of LTD₄ (5R,6S-LTD₄), leukotriene E₄ (LTE₄) and the end-organ antagonist, FPL 55712, we have identified specific binding sites for [³H]-LTD₄ in the crude membrane fraction isolated from guinea pig lung. The time required for [³H]-LTD₄ binding to reach equilibrium was approximately 20 to 25 min at 37°C in the presence of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of [³H]-LTD₄ to the specific sites was saturable, reversible and stereospecific. The maximal number of binding sites (B_max), derived from Scatchard analysis, was approximately 320±200 fmol per mg of crude membrane protein. The dissociation constants, derived from kinetic and saturation analyses, were 9.7 nM and 5±4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand competition experiments, LTD₄, FPL 55712 and 5R,6S-LTD₄ competed with [³H]-LTD₄. The metabolic inhibitors of arachidonic acid and SKF 88046, an antagonist of the indirectly-mediated actions of LTD₄, did not significantly compete with [³H]-LTD₄ at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung.     

Neolignans from an Aniba species

A benzene extract of the trunk wood of an Aniba species contained 3a-allyl-2-aryl-5-methoxy-3-methyl-2,3,3a,6-tetrahydro-6-oxobenzofurans which may be responsible, through sequential Cope, retro-Claisen and Claisen rearrangements respectively for the formation of the co-occurring 5-allyl-2-aryl-5-methoxy-3-methyl-2,3,5,6-tetrahydro-6-oxobenzofurans; the 6-O-allyl-2-aryl-5-methoxy-3-methyl-2,3-dihydrobenzofurans and the 7-allyl-2-aryl-6-hydroxy-5-methoxy-3-methyl-2,3-dihydrobenzofurans. The examination of the stereochemistry of these products led to the formulation of burchellin, previously isolated from Aniba burchellii Kostermans, as (2S,3S,3aR)-3a-allyl-5-methoxy-2-piperonyl-3-methyl-2,3,3a,6-tetrahydro-6-oxobenzofuran. The structure 1-allyl-4,8-dihydroxy-7-(3-methoxy-4,5-methylenedioxyphenyl)-6-methyl-3-oxobicyclo[3,2,1]octane is tentatively proposed for an additional neolignan.     

Receptor-mediated regulation of calcium mobilization and cyclic GMP synthesis in neuroblastoma cells

In neuroblastoma N1E 115 cells, carbachol, histamine and PGE1 elevated cyclic GMP content and, induced the efflux of preloaded 45Ca2+, the release of membrane-bound Ca2+ measured by fluorescent CTC, and the increase in [Ca2+]i as measured by Quin 2 fluorescence. The time course of the responses, the absolute requirement of extracellular Ca2+, the inhibition by receptor blockers, and the concentration dependency on histamine were all similar between these responses. The observation indicates that the mobilization of Ca2+, especially the increase of [Ca2+]i, may be intimately linked to the synthesis of cyclic GMP in the cells.     

A radioimmunoassay method for urinary catechol estrogens

A radioimmunoassay method for urinary catechol estrogens is described. The specific nature of the antisera allows direct analyses of acid hydrolyzed urine. A LH-20 Sephadex column chromatography can be employed for individual determinations of 2-hydroxyestrone and 2-hydroxyestradiol. The excretion of catechol estrogens during menstrual cycles ranged from 14.48 to 50.15 μg per 24 hours, whereas, during the last trimester of pregnancies, the values ranged from 129.30 to 1758.20 μg per 24 hours.     

Theoretical research in structure characteristics of different inhibitors and differences of binding modes with CBP bromodomain

The CBP (CREB (cAMP responsive element binding protein) binding protein) bromodomain (BRD) could recognize and bind with acetyl K382 of human tumor suppressor protein p53 which the mutation of encoding gene might cause human cancers. CBP-BRD serves as a promising drug target for several disease pathways and a series of effective drug have been discovered. In this study, molecular dynamics (MD) simulations and molecular mechanics generalized born surface area (MM-GB/SA) approaches were performed to investigate the different binding modes between five inhibitors with CBP-BRD. Based on the energy and conformation analyses, a potent core fragment is chosen to act as the starting point for new inhibitor design by means of LUDI and rational drug design approaches. Then, T.E.S.T and molinspirition were applied to evaluate oral bioavailability and drug promiscuity of the new molecules. These results shed light on the idea for further inhibitor design.     

Phenolic derivatives from Wigandia urens with weak activity against the chemokine receptor CCR5

Three compounds, 2,3-dihydroxy-4-methoxy-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (1), 8-methoxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzopyran-6-ol (2) and 4-methoxy-3-(3-methyl-2-butenyl)-benzoic acid (3), have been isolated from Wigandia urens. The structures of compounds 1, 2 and 3 were determined from spectroscopic data and showed activity in a CCR5 assay with IC(50) values of 33, 46 and 26 muM respectively.     

Flavones and isoflavones from the west African Fabaceae Erythrina vogelii

The CH(2)Cl(2)/MeOH extract of the stem bark of Erythrina vogelii (Fabaceae) from Nigeria has yielded two novel isoflavones, 7,4'-dihydroxy-8-(gamma,gamma-dimethylallyl)-2'zeta-(4'-hydroxyisopropyl)dihydrofurano[1',3':5,6]isoflavone (vogelin H) (1) and 7,4'-dihydroxy-8-[(2'zeta,3'-dihydroxy-3'-methyl)butyl]-2',2'-dimethyl-3',4'-dehydropyrano[1',4':5,6]isoflavone (vogelin I) (2), a novel flavone, 7,4'-dihydroxy-2',2'-dimethyl-3',4'-dehydropyrano[1',4':5,6]flavone (vogelin J) (3), and eight known flavonoids.     

The many faces of calmodulin in cell proliferation,programmed cell death,autophagy, and cancer

Calmodulin (CaM) is a ubiquitous Ca^2 + receptor protein mediating a large number of signaling processes in all eukaryotic cells. CaM plays a central role in regulating a myriad of cellular functions via interaction with multiple target proteins. This review focuses on the action of CaM and CaM-dependent signaling systems in the control of vertebrate cell proliferation, programmed cell death and autophagy. The significance of CaM and interconnected CaM-regulated systems for the physiology of cancer cells including tumor stem cells, and processes required for tumor progression such as growth, tumor-associated angiogenesis and metastasis are highlighted. Furthermore, the potential targeting of CaM-dependent signaling processes for therapeutic use is discussed.     

Systematics and health effects of chemically distinct tannins in medicinal plants

The research began with an investigation of tannins from traditional medicinal plants and resulted in isolation and structure determination of hundreds of ellagitannins and dehydroellagitannins, as well as their oligomers and oxidized derivatives with various structures specific to each plant species. These polyphenols have been classified according to the stage of oxidative structural transformation and oligomerization, into types I-IV and I+ to IV+, etc. Parallels were found between their oxidative transformations and plant evolution. They were also classified by the linkage units between the monomers, into DOG, GOD, GOG and DOGOD types (D=Diphenoyl, G=Galloyl, O=Oxygen), etc. Besides their fundamental activities, e.g., reduction and anti-peroxidation properties, remarkable biological and pharmacological activities of various potencies have also been found, including, amongst others, inhibition of lipid-peroxidation, mutagenicity of carcinogens and tumor promotion, host-mediated antitumor effects specific to particular tannin structures, antiviral activity and potentiation of antibacterial activity.     

The astrocyte excitability brief: from receptors to gliotransmission

Astrocytes do not merely serve as the supporting cast and scenery against which starring roles would be played by neurons. Rather, these glial cells are intimately involved in many of the brain's functions by responding to neuronal activity and modulating it. Such interplay between two principle neural cells, neurons and astrocytes, is evidenced in bi-directional glutamatergic astrocyte-neuron signaling. A key feature in this signaling pathway is astrocytic excitability based on variations of cytosolic Ca(2+). It enables astrocytes, through the activation of their glutamatergic receptors, to respond to the same signal used by nearby neurons in synaptic transmission. Furthermore, increases in cytosolic Ca(2+) in astrocytes can subsequently lead to Ca(2+)-dependent exocytotic secretion of gliotransmitter glutamate that in turn can signal to adjacent neurons. Astrocytic secretory machinery includes an assortment of exocytotic proteins which governs a merger of secretory vesicles to the plasma membrane. A cumulative knowledge on astrocytic excitability will aid better understanding of operating procedures in the brain in health and disease.     

Different mechanisms involved in apoptosis following exposure to benzo[a]pyrene in F258 and Hepa1c1c7 cells

The present study compares and elucidates possible mechanisms why B[a]P induces different cell signals and triggers apparently different apoptotic pathways in two rather similar cell lines (hepatic epithelial cells of rodents). The rate and maximal capacity of metabolic activation, as measured by the formation of B[a]P-tetrols and B[a]P-DNA adducts, was much higher in mouse hepatoma Hepa1c1c7 cells than in rat liver epithelial F258 cells due to a higher induced level of cyp1a1. B[a]P increased intracellular pH in both cell lines, but this change modulated the apoptotic process only in F258 cells. In Hepa1c1c7 cells reactive oxygen species (ROS) production appeared to be a consequence of toxicity, unlike F258 cells in which it was an initial event. The increased mitochondrial membrane potential found in F258 cells was not observed in Hepa1c1c7 cells. Surprisingly, F258 cells cultured at low cell density were somewhat more sensitive to low (50nM) B[a]P concentrations than Hepa1c1c7 cells. This could be explained partly by metabolic differences at low B[a]P concentrations. In contrast to the Hepa1c1c7 model, no activation of cell survival signals including p-Akt, p-ERK1/2 and no clear inactivation of pro-apoptotic Bad was observed in the F258 model following exposure to B[a]P. Another important difference between the two cell lines was related to the role of Bax and cytochrome c. In Hepa1c1c7 cells, B[a]P exposure resulted in a "classical" translocation of Bax to the mitochondria and release of cytochrome c, whereas in F258 cells no intracellular translocation of these two proteins was seen. These results suggest that the rate of metabolism of B[a]P and type of reactive metabolites formed influence the resulting balance of pro-apoptotic and anti-apoptotic cell signaling, and hence the mechanisms involved in cell death and the chances of more permanent genetic damage.     

Pharmacological modulation of NMDA receptor activity and the advent of negative and positive allosteric modulators

The NMDA receptor (NMDAR) family of l-glutamate receptors are well known to have diverse roles in CNS function as well as in various neuropathological and psychiatric conditions. Until recently, the types of agents available to pharmacologically regulate NMDAR function have been quite limited in terms of mechanism of action and subtype selectivity. This has changed significantly in the past two years. The purpose of this review is to summarize the many drug classes now available for modulating NMDAR activity. Previously, this included competitive antagonists at the l-glutamate and glycine binding sites, high and low affinity channel blockers, and GluN2B-selective N-terminal domain binding site antagonists. More recently, we and others have identified new classes of NMDAR agents that are either positive or negative allosteric modulators (PAMs and NAMs, respectively). These compounds include the pan potentiator UBP646, the GluN2A-selective potentiator/GluN2C and GluN2D inhibitor UBP512, the GluN2D-selective potentiator UBP551, the GluN2C/GluN2D-selective potentiator CIQ as well as the new NMDAR-NAMs such as the pan-inhibitor UBP618, the GluN2C/GluN2D-selective inhibitor QZN46 and the GluN2A inhibitors UBP608 and TCN201. These new agents do not bind within the l-glutamate or glycine binding sites, the ion channel pore or the N-terminal regulatory domain. Collectively, these new allosteric modulators appear to be acting at multiple novel sites on the NMDAR complex. Importantly, these agents display improved subtype-selectivity and as NMDAR PAMs and NAMs, they represent a new generation of potential NMDAR therapeutics.     

Role of S-adenosylhomocysteine hydrolase in adenosine-induced apoptosis in HepG2 cells

Adenosine has been shown to initiate apoptosis through different mechanisms: (i) activation of adenosine receptors, (ii) intracellular conversion to AMP and stimulation of AMP-activated kinase, (iii) conversion to S-adenosylhomocysteine (AdoHcy), which is an inhibitor of S-adenosylmethionine (AdoMet)-dependent methyltransferases. Since the pathways involved are still not completely understood, we further investigated the role of AdoHcy hydrolase in adenosine-induced apoptosis. In HepG2 cells, adenosine induced caspase-like activity and DNA fragmentation, a marker of apoptosis. These effects were potentiated by co-incubation with homocysteine or adenosine deaminase inhibitor, pentostatin, and were mimicked by inhibition of AdoHcy hydrolase by adenosine-2',3'-dialdehyde (Adox). Adenosine-induced effects were significantly inhibited by dipyridamole, an inhibitor of adenosine transporter, whereas inhibitors of adenosine kinase did not affect adenosine-induced changes. Various adenosine receptor agonists and AICAR, an activator of AMP-activated kinase, did not mimic the effect of adenosine. Thus, adenosine-induced apoptosis is likely due to intracellular action of AdoHcy and independent of AMP-activated kinase and adenosine receptors. Because elevated AdoHcy levels are associated with reduced mRNA methylation, we studied mRNA expression in Adox-treated cells by microarray analysis. Since several p53-target genes and other apoptosis-related genes were up-regulated by Adox, we conclude that AdoHcy is involved in adenosine-induced apoptosis by altering gene expression.     

Lipid and lipid mediator profiling of human synovial fluid in rheumatoid arthritis patients by means of LC–MS/MS

Human synovial fluid (SF) provides nutrition and lubrication to the articular cartilage. Particularly in arthritic diseases, SF is extensively accumulating in the synovial junction. During the last decade lipids have attracted considerable attention as their role in the development and resolution of diseases became increasingly recognized. Here, we describe a capillary LC–MS/MS screening platform that was used for the untargeted screening of lipids present in human SF of rheumatoid arthritis (RA) patients. Using this platform we give a detailed overview of the lipids and lipid‐derived mediators present in the SF of RA patients. Almost 70 different lipid components from distinct lipid classes were identified and quantification was achieved for the lysophosphatidylcholine and phosphatidylcholine species. In addition, we describe a targeted LC–MS/MS lipid mediator metabolomics strategy for the detection, identification and quantification of maresin 1, lipoxin A₄ and resolvin D5 in SF from RA patients. Additionally, we present the identification of 5S,12S-diHETE as a major marker of lipoxygenase pathway interactions in the investigated SF samples. These results are the first to provide a comprehensive approach to the identification and profiling of lipids and lipid mediators present in SF and to describe the presence of key anti-inflammatory and pro-resolving lipid mediators identified in SF from RA patients.     

Three decades of nucleic acid aptamer technologies: Lessons learned,progress and opportunities on aptamer development

Aptamers are short single-stranded nucleic acid sequences capable of binding to target molecules in a way similar to antibodies. Due to various advantages such as prolonged shelf life, low batch to batch variation, low/no immunogenicity, freedom to incorporate chemical modification for enhanced stability and targeting capacity, aptamers quickly found their potential in diverse applications ranging from therapy, drug delivery, diagnosis, and functional genomics to bio-sensing. Aptamers are generated by a process called SELEX. However, the current overall success rate of SELEX is far from being satisfactory, and still presents a major obstacle for aptamer-based research and application. The need for an efficient selection strategy consisting of defined procedures to deal with a wide variety of targets is significantly important. In this work, by analyzing key aspects of SELEX including initial library design, target preparation, PCR optimization, and single strand DNA separation, we provide a comprehensive analysis of individual steps to facilitate researchers intending to develop personalized protocols to address many of the obstacles in SELEX. In addition, this review provides suggestions and opinions for future aptamer development procedures to address the concerns on key SELEX steps, and post-SELEX modifications.     

Successful amphiphiles as the key to crystallization of membrane proteins: Bridging theory and practice

Background

Membrane proteins constitute a major group of proteins and are of great significance as pharmaceutical targets, but underrepresented in the Protein Data Bank. Particular reasons are their low expression yields and the constant need for cautious and diligent handling in a sufficiently stable hydrophobic environment substituting for the native membrane. When it comes to protein crystallization, such an environment is often established by detergents.

Exemestane metabolites suppress growth of estrogen receptor-positive breast cancer cells by inducing apoptosis and autophagy: A comparative study with Exemestane

Around 60–80% of all breast tumors are estrogen receptor-positive. One of the several therapeutic approaches used for this type of cancers is the use of aromatase inhibitors. Exemestane is a third-generation steroidal aromatase inhibitor that undergoes a complex and extensive metabolism, being catalytically converted into chemically active metabolites. Recently, our group showed that the major exemestane metabolites, 17β-hydroxy-6-methylenandrosta-1,4-dien-3-one and 6-(hydroxymethyl)androsta-1,4,6-triene-3,17-dione, as well as, the intermediary metabolite 6β-Spirooxiranandrosta-1,4-diene-3,17-dione, are potent aromatase inhibitors in breast cancer cells. In this work, in order to better understand the biological mechanisms of exemestane in breast cancer and the effectiveness of its metabolites, it was investigated their effects in sensitive and acquired-resistant estrogen receptor-positive breast cancer cells. Our results indicate that metabolites induced, in sensitive breast cancer cells, cell cycle arrest and apoptosis via mitochondrial pathway, involving caspase-8 activation. Moreover, metabolites also induced autophagy as a promoter mechanism of apoptosis. In addition, it was demonstrated that metabolites can sensitize aromatase inhibitors-resistant cancer cells, by inducing apoptosis. Therefore, this study indicates that exemestane after metabolization originates active metabolites that suppress the growth of sensitive and resistant breast cancer cells. It was also concluded that, in both cell lines, the biological effects of metabolites are different from the ones of exemestane, which suggests that exemestane efficacy in breast cancer treatment may also be dependent on its metabolites.