Coumarin A/AA induces apoptosis‐like cell death in HeLa cells mediated by the release of apoptosis‐inducing factor

It has been demonstrated that naturally occurring coumarins have strong biological activity against many cancer cell lines. In this study, we assessed the cytotoxicity induced by the naturally isolated coumarin A/AA in different cancer cell lines (HeLa, Calo, SW480, and SW620) and in normal peripheral‐blood mononuclear cells (PBMCs). Cytotoxicity was evaluated using the MTT assay. The results demonstrate that coumarin A/AA was cytotoxic in the four cancer cell lines tested and importantly was significantly less toxic in PBMCs isolated from healthy donors. The most sensitive cancer cell line to coumarin A/AA treatment was Hela. Thus, the programmed cell death (PCD) mechanism induced by this coumarin was further studied in this cell line. DNA fragmentation, histomorphology, cell cycle phases, and subcellular distribution of PCD proteins were assessed. The results demonstrated that DNA fragmentation, but not significant cell cycle disruptions, was part of the PCD activated by coumarin A/AA. Interestingly, it was found that apoptosis‐inducing factor (AIF), a proapoptotic protein of the mitochondrial intermembrane space, was released to the cytoplasm in treated cells as detected by the western blot analysis in subcellular fractions. Nevertheless, the active form of caspase‐3 was not detected. The overall results indicate that coumarin A/AA induces a caspase‐independent apoptotic‐like cell death program in HeLa cells, mediated by the early release of AIF and suggest that this compound may be helpful in clinical oncology. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:263–272, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20288     

Down-regulation of anandamide hydrolase in mouse uterus by sex hormones.

Endocannabinoids are an emerging class of lipid mediators, which mimic several effects of cannabinoids. Anandamide (arachidonoylethanolamide) is a major endocannabinoid, which has been shown to impair pregnancy and embryo development. The activity of anandamide is controlled by cellular uptake through a specific transporter and intracellular degradation by the enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH). We characterized FAAH in mouse uterus by radiochromatographic and immunochemical techniques, showing that the enzyme is confined to the epithelium and its activity decreases appreciably during pregnancy or pseudopregnancy because of lower gene expression at the translational level. Ovariectomy prevented the decrease in FAAH, and both progesterone and estrogen further reduced its basal levels, suggesting hormonal control of the enzyme. Anandamide was shown to induce programmed cell death in mouse blastocysts, through a pathway independent of type-1 cannabinoid receptor. Blastocysts, however, have a specific anandamide transporter and FAAH, which scavenge this lipid. Taken together, these results provide evidence of an interplay between endocannabinoids and sex hormones in pregnancy. These findings may also be relevant for human fertility, as epithelial cells from healthy human uterus showed FAAH activity and expression, which in adenocarcinoma cells was increased fivefold.     

Unravelling Glucan Recognition Systems by Glycome Microarrays Using the Designer Approach and Mass Spectrometry

Glucans are polymers of d-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes, including immunomodulation, anticancer activities, pathogen virulence, and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure–function studies and their exploitation. We describe construction of a “glucome” microarray, the first sequence-defined glycome-scale microarray, using a “designer” approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear “homo” and “hetero” and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.Glucan polysaccharides are polymers of d-glucose with differing linkages in linear or branched sequences. They occur as storage materials in animals, secreted virulence factors of bacteria, and conserved structural components of cell walls of yeasts, fungi, some bacteria, and plants. Polysaccharides of this type are of considerable interest in biology, medicine, and biotechnology and are acknowledged for their immunostimulatory, anticancer, and health-promoting activities (1, 2); for their elicitor activities in defense responses and signaling in plants (3); and for acting as functional ingredients in human nutrition (4). Unraveling recognition systems that mediate these activities is highly desirable as a lead to effective translational applications.Recognition systems involving glucan polysaccharides include those in mammals, such as recognition of fungal β-glucans by Dectin-1, the major receptor of the innate immune system against fungal pathogens (5), and by natural or vaccine-induced protective antifungal antibodies (6, 7); also recognition of mycobacterial α-glucan by the innate immune receptor DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin) (8); those in insects, such as the Drosophila Gram-negative binding protein 3 (GNBP3) sensor protein, which binds β-glucans (9); and those in bacteria, such as Brucella abortus, where cyclic β-glucans can serve as virulence factors (10).Another important class of glucan-recognizing proteins comprises noncatalytic carbohydrate-binding modules (CBMs)¹ of bacterial glycoside hydrolases that mediate association with substrate and increase catalytic activity, likely through a targeting mechanism or by driving enzyme specificity (11, 12). Notable examples are CBMs of bacterial cellulolytic enzymes that promote enzymatic deconstruction of intact plant cell walls and that are of industrial significance in the biofuel and bioprocessing sectors (13, 14) and CBMs of rumen or commensal human microbiota with roles in animal and human health (14, 15). CBMs also have roles in other systems: for example, CBM-containing enzymes as virulence factors of bacterial pathogens (16) and CBM-containing human laforin that regulates glycogen metabolism and for which mutations can lead to neurodegenerative disease (17). The number of putative glucan-binding CBMs that have been identified and classified in the Carbohydrate-Active enZyme (CAZy) database (http://www.cazy.org) is expanding, but relatively few have been experimentally investigated for details of carbohydrate binding and fine specificity (11).Searching for and assigning the specificities of glucan-recognizing proteins has thus become increasingly important. It is desirable to have high-throughput and sensitive micro-methods to screen for and characterize ligands for structure–function studies toward effective exploitation in modern therapeutic, nutritional, agricultural, and biofuel-related technologies. Carbohydrate microarrays have served to advance knowledge on specificities of diverse carbohydrate-recognition systems (18–22). Where the desired oligosaccharide probes are unavailable, microarrays need to be generated from ligand-bearing glycomes (23). Using a prototype of such designer microarrays of neoglycolipid (NGL)-probes (23) derived from oligosaccharide fragments of glucans rich in β1,3- or β1,6-linked sequences, we showed that linear β1,3-linked glucose sequences with degree of polymerization (DP) 10 or longer are bound by Dectin-1 (24). Recognition of other types of glucan sequences by Dectin-1 and the applicability of microarrays of diverse gluco-oligosaccharide sequences to other glucan-recognizing proteins required investigation. Cummings, Smith, and colleagues have developed the shotgun strategy (20) to create glycome-scale “gangliome” and “human milk glycome” microarrays. In the shotgun microarrays, the printed probes may not be sequence-defined before array construction and require metadata-assisted glycan sequencing (MAGS), which combines MS analysis (25), binding data with glycan-binding proteins or antibodies, and exoglycosidase treatment after printing (26, 27).Mass spectrometry has become a primary technique in carbohydrate structural analysis (28), and electrospray mass spectrometry (ESI-MS) has been used to provide sequence and partial linkage information on various types of oligosaccharides (29–33). For neutral oligosaccharides, we have found that tandem MS with collision-induced dissociation (CID-MS/MS) in the negative-ion mode is particularly useful and have successfully applied for oligosaccharide chain and blood-group typing (34, 35) and for branching pattern analysis (36).This is because that some important linkages at certain monosaccharide residues can be unambiguously determined with high sensitivity without the need for derivatization and anion complexation as previously recognized, e.g. in the area of gluco-oligosaccharides, Cl⁻-anion adduction has been used to determine sequences of tetrasaccharides of dextran (37).Here, we describe a strategy using the designer approach combined with negative-ion ESI-CID-MS/MS for constructing a microarray of sequence-defined gluco-oligosaccharides representing major sequences in glucans (glucome microarray) as a tool for screening glucan-recognizing proteins and assigning their recognition motifs (Fig. 1). We selected a comprehensive panel of glucan polysaccharides isolated from plants, fungi, and bacteria with different sequences to represent the glucome. We used finely tuned chemical and enzymatic methods to partially depolymerize the polysaccharides and prepare gluco-oligosaccharide fragments with different chain lengths (up to DP-13 or DP-16). We developed a ESI-CID-MS/MS method that enables linkage and sequence determination of linear or branched gluco-oligosaccharides at high-sensitivity and applied this to the sequencing of oligosaccharide fragments prepared. These sequence-defined gluco-oligosaccharides were then converted into NGL probes and used for construction of the microarray. The oligosaccharides encompassed linear sequences with homo (single) linkages: 1,2-, 1,3-, 1,4-, or 1,6- with α or β configurations; and hetero (multiple) linkages: 1,3-, 1,4, or 1,6-; also branched oligosaccharide sequences with 1,3 and 1,6-linkages.Open in a separate windowFig. 1.Neoglycolipid (NGL)-based designer glucome microarray with mass spectrometry as a tool to assign carbohydrate ligands in glucan recognition. Ligand-bearing glucan polysaccharides, described in supplemental Fig. S1 and Table S1, were selected as sources of gluco-oligosaccharides for construction of the microarray. A total of 121 gluco-oligosaccharide fractions were obtained with different DP after partial depolymerization of polysaccharides and fractionation. ESI-CID-MS/MS method was developed using gluco-oligosaccharides with known sequences and applied to determination of sequences of oligosaccharide fragments from polysaccharides. Gluco-oligosaccharides were converted to NGL probes for microarray construction and interrogation with the glucan-recognizing proteins described in supplemental Table S2.To our knowledge, this is the first sequence-defined glycome-scale microarray constructed. We used 12 selected proteins (antibodies and CBMs) known to target α- or β-glucans to validate the approach. We then applied the microarray analysis to Dectin-1 and DC-SIGN, which revealed new insights into the specificities of these signaling molecules of the innate immune system.     

Genome-Wide Association Study of Late-Onset Myasthenia Gravis: Confirmation of TNFRSF11A and Identification of ZBTB10 and Three Distinct HLA Associations

To investigate the genetics of late-onset myasthenia gravis (LOMG), we conducted a genome-wide association study imputation of >6 million single nucleotide polymorphisms (SNPs) in 532 LOMG cases (anti–acetylcholine receptor [AChR] antibody positive; onset age ≥50 years) and 2,128 controls matched for sex and population substructure. The data confirm reported TNFRSF11A associations (rs4574025, P = 3.9 × 10⁻⁷, odds ratio [OR] 1.42) and identify a novel candidate gene, ZBTB10, achieving genome-wide significance (rs6998967, P = 8.9 × 10⁻¹⁰, OR 0.53). Several other SNPs showed suggestive significance including rs2476601 (P = 6.5 × 10⁻⁶, OR 1.62) encoding the PTPN22 R620W variant noted in early-onset myasthenia gravis (EOMG) and other autoimmune diseases. In contrast, EOMG-associated SNPs in TNIP1 showed no association in LOMG, nor did other loci suggested for EOMG. Many SNPs within the major histocompatibility complex (MHC) region showed strong associations in LOMG, but with smaller effect sizes than in EOMG (highest OR ~2 versus ~6 in EOMG). Moreover, the strongest associations were in opposite directions from EOMG, including an OR of 0.54 for DQA1*05:01 in LOMG (P = 5.9 × 10⁻¹²) versus 2.82 in EOMG (P = 3.86 × 10⁻⁴⁵). Association and conditioning studies for the MHC region showed three distinct and largely independent association peaks for LOMG corresponding to (a) MHC class II (highest attenuation when conditioning on DQA1), (b) HLA-A and (c) MHC class III SNPs. Conditioning studies of human leukocyte antigen (HLA) amino acid residues also suggest potential functional correlates. Together, these findings emphasize the value of subgrouping myasthenia gravis patients for clinical and basic investigations and imply distinct predisposing mechanisms in LOMG.     

Draft Genome Sequence of the Volatile Organic Compound-Producing Antarctic Bacterium Arthrobacter sp. Strain TB23, Able To Inhibit Cystic Fibrosis Pathogens Belonging to the Burkholderia cepacia Complex

Arthrobacter sp. strain TB23 was isolated from the Antarctic sponge Lissodendoryx nobilis. This bacterium is able to produce antimicrobial compounds and volatile organic compounds (VOCs) that inhibit the growth of other Antarctic bacteria and of cystic fibrosis opportunistic pathogens, respectively. Here we report the draft genome sequence of Arthrobacter sp. TB23.     

An assessment of natural and human disturbance effects on Mexican ecosystems: current trends and research gaps

Mexico harbors more than 10% of the planet’s endemic species. However, the integrity and biodiversity of many ecosystems is experiencing rapid transformation under the influence of a wide array of human and natural disturbances. In order to disentangle the effects of human and natural disturbance regimes at different spatial and temporal scales, we selected six terrestrial (temperate montane forests, montane cloud forests, tropical rain forests, tropical semi-deciduous forests, tropical dry forests, and deserts) and four aquatic (coral reefs, mangrove forests, kelp forests and saline lakes) ecosystems. We used semi-quantitative statistical methods to assess (1) the most important agents of disturbance affecting the ecosystems, (2) the vulnerability of each ecosystem to anthropogenic and natural disturbance, and (3) the differences in ecosystem disturbance regimes and their resilience. Our analysis indicates a significant variation in ecological responses, recovery capacity, and resilience among ecosystems. The constant and widespread presence of human impacts on both terrestrial and aquatic ecosystems is reflected either in reduced area coverage for most systems, or reduced productivity and biodiversity, particularly in the case of fragile ecosystems (e.g., rain forests, coral reefs). In all cases, the interaction between historical human impacts and episodic high intensity natural disturbance (e.g., hurricanes, fires) has triggered a reduction in species diversity and induced significant changes in habitat distribution or species dominance. The lack of monitoring programs assessing before/after effects of major disturbances in Mexico is one of the major limitations to quantifying the commonalities and differences of disturbance effects on ecosystem properties.     

Ligands for the beta-glucan receptor, Dectin-1, assigned using "designer" microarrays of oligosaccharide probes (neoglycolipids) generated from glucan polysaccharides

Dectin-1 is a C-type lectin-like receptor on leukocytes that mediates phagocytosis and inflammatory mediator production in innate immunity to fungal pathogens. Dectin-1 lacks residues involved in calcium ligation that mediates carbohydrate-binding by classical C-type lectins; nevertheless, it binds zymosan, a particulate beta-glucan-rich extract of Saccharomyces cerevisiae, and binding is inhibited by polysaccharides rich in beta1,3- or both beta1,3- and beta1,6-linked glucose. The oligosaccharide ligands on glucans recognized by Dectin-1 have not yet been delineated precisely. It is also not known whether Dectin-1 can interact with other types of carbohydrates. We have investigated this, since Dectin-1 shows glucan-independent binding to a subset of T-lymphocytes and is involved in triggering their proliferation. Here we assign oligosaccharide ligands for Dectin-1 using the neoglycolipid-based oligosaccharide microarray technology, a unique approach for constructing microarrays of lipid-linked oligosaccharide probes from desired sources. We generate "designer" microarrays from three glucan polysaccharides, a neutral soluble glucan isolated from S. cerevisiae and two bacterial glucans, curdlan from Alcaligenes faecalis and pustulan from Umbilicaria papullosa, and use these in conjunction with 187 diverse, sequence-defined, predominantly mammalian-type, oligosaccharide probes. Among these, Dectin-1 binding is detected exclusively to 1,3-linked glucose oligomers, the minimum length required for detectable binding being a 10- or 11-mer. Thus, the ligands assigned so far are exogenous rather than endogenous. We further show that Dectin-1 ligands, 11-13 gluco-oligomers, in clustered form (displayed on liposomes), mimic the macromolecular beta-glucans and compete with zymosan binding and triggering of tumor necrosis factor-alpha secretion by a Dectin-1-expressing macrophage cell line.     

Expedited SAR study of high-affinity ligands to the alpha(2)delta subunit of voltage-gated calcium channels: Generation of a focused library using a solution-phase Sn2Ar coupling methodology

The SAR of the lead compound 3, a novel ligand for the alpha(2)delta subunit of voltage-gated calcium channels, was rapidly explored. Utilizing a parallel solution-phase Sn2Ar coupling approach, a focused library was obtained. The library was evaluated in vitro and afforded a series of analogues with improved potencies. The SAR trends of the library are also described.