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3 von 4 Kunden fanden die folgende Rezension hilfreich
The Mastery and Mystery of Bread Illuminated,
Rezension bezieht sich auf: Crust and Crumb: Master Formulas for Serious Bread Bakers (Gebundene Ausgabe)
I purchased Crust & Crumb as another addition to my many cookbooks on bread baking. I sat down to scan my new purchase and the next thing I realized is it was 2:00am and I'd just read the book word for word, cover to cover. I honestly believe I could throw out the majority of my books on bread baking and not ever miss them as long as Crust & Crumb was on my shelf. One telltale sign of the books influence on myself is that my Kitchenaid Mixer is gathering dust as I roll up my sleeves and passionatly begin kneading. Once again connecting to the magic of transforming singular ingredients into a living, united mystery that is more than the sum of it's parts. I feel I've found a kinderd spirit in Brother Peter, and am truly inspired by his knowledge of the history about bread, the fundamental meaning of bread to mankind and the humble passion he has for the process. Brother Peter walks one through the combining of the age old flour, water and salt, and then encourages ones "unbeilf" as grace takes over and a golden, crusty loaf is born. As spacey as these musings sound, the book is concrete, practical and it follows,easy step by step formulas. I try very hard to find the meaning and mystery in the everyday (mundane) events such as baking bread. Brother Peter has done that for us in "Crust & Crumb".
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Carbohydrates and carbohydrate-containing biomolecules engage in binding events that underlie many essential biological processes. Yet these carbohydrate-mediated interactions are often poorly characterized, due to their low affinities and heterogenous natures. The use of photocrosslinking functional groups offers a way to photochemically capture carbohydrate-containing complexes, which can be isolated for further analysis. Here we survey progress in the synthesis and use of carbohydrate-based photoprobes, reagents that incorporate carbohydrates or their analogs, photocrosslinking moieties, and affinity purification handles. Carbohydrate photoprobes, used in combination with modern mass spectrometry methods, can provide important new insights into the cellular roles of carbohydrates and glycosylated molecules.
Carbohydrates are key mediators of biological interactions. The glycan components of glycoproteins and glycolipids are critical to the function of these molecules and regulate their ability to interact with binding partners. The interactions of glycosylated molecules play essential roles in diverse areas of biology, notably in immunology, developmental processes, and infectious disease. Despite their essential roles, glycan-mediated interactions are typically low-affinity, often displaying equilibrium dissociation constants in the micromolar or even millimolar range. Thus, isolating and defining the binding partners that engage in glycan-mediated binding interactions is challenging because the labile complexes often fail to withstand the necessary purification steps. One possible solution to this challenge is to use photocrosslinking reagents to introduce a covalent bond between the binding partners. Once covalently constrained, complexes can be isolated and the binding partners characterized using routine analytical methods.
Photocrosslinking functional groups must possess certain key features in order to function in the capture of biologically relevant interactions. These functional groups must be bioorthogonal to cellular functional groups, so that they do not engage in spurious reactions. They should be stable until activated by application of light of an appropriate wavelength. Ideally, photocrosslinking groups will be excited by a long wavelength of light, to minimize cellular damage, and should have a high crosslinking efficiency, with minimal propensity for side reactions. Finally, the size of the photocrosslinking group is important, as larger functional groups have the potential to sterically interfere with the interactions under study. To date, four classes of photocrosslinking groups have been widely used in biological settings: benzophenones, aryl azides, alkyl diazirines, and trifluoromethyl phenyl diazirines. Further discussion of the relative merits and weaknesses of these photocrosslinking groups can be found elsewhere.[1]
This review article describes a variety of photoactivatable probes that have been prepared and used to study carbohydrate biology. Each of the probes contains some type of targeting feature, typically a glycan or glycan analog, that drives association with the carbohydrate-binding pocket of an enzyme or glycan-binding protein. In the figures shown in this article, these interaction motifs are depicted in blue. Each photoactivable probe also contains a light-activated photocrosslinking group, typically a benzophenone, aryl azide, or diazirine, that enables the probe to form covalent bonds with the targeted enzyme or protein. In the figures shown in this article, photocrosslinking groups are diagrammed in red. Many, although not all, of the probes discussed here also include handles that may be used for detection (radiolabels, fluorophores) or affinity purification (biotin, alkynes, azides); these functionalities are indicated in green.
Glycoside hydrolases, or glycosidases, are hydrolytic enzymes that cleave glycosidic bonds. Constituting a large enzyme family found in all domains of life, glycosidases vary in glycoconjugate specificity and function.[2–4] Glycosidases play essential roles in biomass degradation, nutrient acquisition, viral and bacterial pathogenesis, and host defense. They also catalyze key steps required in glycoconjugate biosynthesis (for example, N-linked glycan assembly). As described below, photoaffinity probes have potential applications in defining the active sites of glycosidases and in isolating glycosidases with particular glycan specificities from complex mixtures.
Achieving a specific reaction between a small molecule probe and a glycosidase active site is challenging because of the transient nature of glycosidase-substrate binding interactions. In some cases, enzymatically-activated probes, which generate reactive species such as quinone methide, have successfully been used to covalently label the active sites of glycosidases.[5] One intrinsic disadvantage with the use of these latently reaction probes is that, upon activation, the targeting reagent and the reactive species separate, resulting in the potential for non-specific labeling. Mechanism-based inactivating probes based on fluorosugars[6,7] are another powerful approach, although their utility can be limited by slow reaction kinetics or incomplete knowledge of reaction mechanism. Photocrosslinking probes offer another alternative to the existing approaches. Since the recognition (carbohydrate) element remains attached to the photoreactive moiety throughout the crosslinking process, photocrosslinking probes are expected to result in less non-specific labeling than enzymatically-activated probes. In addition, once photoactivated, photocrosslinking probes have very rapid reaction kinetics, so high efficiency labeling can be achieved. A limitation of most photocrosslinking probes is that they are not enzyme-activated, so they may also label glycan-binding proteins, rather than being selective for glycosidases.
Below, we describe examples of photocrosslinking reagents designed to selectively label glycosidases, and the utility of these reagents in defining enzyme active sites and in isolating glycosidases with specificity for particular glycans. Key features of these reagents include the enzyme-binding motifs, typically glycans, the selection of photoreactive functional group, and the choice of labeling or affinity probes. In many cases, glycosidases have tightly-packed active sites that do not allow space for the affinity label. Thus, ideal photoaffinity group and reporter group placement through a well-designed linker are important factors in the successful use of these reagents.
Orth et al. reported a modular synthesis of photoprobes designed for labeling and affinity purification of bacterial glycosidases.[8] Using the 1,3-dipolar Huisgen cycloaddition reaction, they coupled β-azidosugars (glucose, galactose, or N-acetylglucosamine) through a non-hydrolyzable linkage (triazole) to a peptide linker modified with a photocrosslinker (benzophenone) and an alkyne handle to be used for subsequent isolation (Figure 1A). Using these three carbohydrate probes, the researchers confirmed the glycan specificity of recombinant NagZ, a β-N-acetylglucosaminidase from E. coli: only the N-acetylglucosamine probe was efficiently crosslinked to the enzyme. While this initial report did not go on to investigate whether the photoprobes can be used in a more complex mixture for glycosidase isolation and identification, the modular route by which these reagents were assembled could be generally useful since complex carbohydrates with an azido functional group at the reducing end are readily available and could be assembled to the similar type of photoprobes
Glucosidases, enzymes that hydrolytically release glucose residues, are found in a variety of organisms and play diverse functional roles. For example, in mammals, glucosidase I is a regulator of N-linked glycan maturation, while lysosomal β-glucosidase catalyzes an essential step in glucosylceramide degradation. Termites and some microorganisms rely on glucosidases to degrade the cellulose components of plant cell walls. 1-Deoxynojirimycin, an iminosugar initially discovered in the root bark of mulberry tree,[9] and its N-alkyl derivatives are potent glucosidase inhibitors. As a non-hydrolyzable analog of glucosidase substrates, 1-deoxynojirimycin functions as an excellent affinity probe for glucosidases. In 2004, Romaniouk et al. synthesized a probe that contained deoxynojirimycin conjugated to an aryl azide photocrosslinker with an 125I label (Figure 1B). In an experiment conducted in bovine mammary gland extract, this reagent enabled selective labeling of a 24 kDa peptide containing the potential active site of glucosidase I.[10] Also, consistent with the inhibitory activity of 1-deoxynojirimycin, this probe showed selective labeling of glucosidase I over glucosidase II. While the crosslinked complex could be visualized by autoradiography, the radiolabeled probe did not have an affinity handle to enable isolation and identification of the crosslinked species. More recently, Pieters's group reported another deoxynojirimycin-based probe containing both a photocrosslinking functionality and handle for fluorescent labeling.[11] These probes were prepared by conjugating propargylated photocrosslinkers to 1-deoxynojirimycin or glucose; the propargyl group was employed for fluorescent labeling through a 1,3-dipolar cycloaddition with a fluorescein-azide adduct. The benzophenone-conjugated deoxynojirimycin (Figure 1C) yielded efficient labeling of a lysosomal glucocerebrosidase in the presence of HeLa cell lysate, while a glucose analog suffered from poorly labeling efficiency, likely because the probe was hydrolyzed by the enzyme. Taken together, these results demonstrate that sugar analogs can provide useful starting points for photoaffinity probes.
One important application for glycosidase probes is to isolate and identify glycosidases present in complex mixtures. To achieve this, probes must achieve efficient and selective enzyme labeling, as described above, but also contain an affinity tag that enables effective purification of crosslinked species. With the goal of identifying exoglycosidases, glycosidases that remove terminal monsaccharides, Gandy et al. prepared a series of iminosugar-based photocrosslinkable probes.[12] They synthesized these probes by coupling 1-deoxynojirimycin, 1-deoxymannozirimycin, or 1-deoxygalactonojirimycin to a bifunctional azidomethyl aryl azide (Figure 1D). These probes could be first photocrosslinked to a target glycosidase through the aryl azide and then conjugated, through Staudinger ligation, to a phosphine-FLAG reagent, to be used for subsequent isolation of the crosslinked complex using an anti-FLAG antibody. Model reactions with probes based on each of the three iminosugars demonstrated successful crosslinking to purified enzymes. Furthermore, the 1-deoxynojirimycinbased reagent was successfully used to isolate two proteins from a bacterial lysate. The molecular weights of the isolated species were consistent with known exo-α-glycosidases of the source bacterium, although further verification of identity of the protein bands was not described in this initial report.
There are relatively few reports of the use of photocrosslinking reagents to study enzymes involved in carbohydrate metabolism. In one recent example, Huh et al. reported use of [32P]5-azido-UDP-glucose (Figure 2A) to identify the UDP-glucose binding site of human UDP-glucose dehydrogenase (UGDH).[13] This approach employed a compact photocrosslinking probe, in which the modified base, 5-azidouridine, functioned as the photocrosslinking moiety, while the phosphates provided an affinity purification handle. The 32P radiolabel provided an additional reporter to track the formation and isolation of the crosslinked adduct. After photoaffinity labeling of recombinant human UGDH with [32P]5-azido-UDP-glucose and subsequent tryptic digest of the enzyme, a labeled peptide was isolated through phosphate-based affinity capture. The site of photoaffinity labeling was determined by sequencing the peptide and confirmed by additional experiments conducted with point mutants of UGDH. This elegant use of a modified nucleotide sugar provided new information about the ligand-binding site of UGDH; however, the approach is likely limited to cases where purified enzyme is available since complex mixtures will contain other many other phosphopeptides that would complicate analysis.
Glycosyltransferases are enzymes that form new glycosidic bonds by transferring monosaccharides from activated nucleotide sugar donors, such as UDP-glucose, to acceptor molecules. Glycosyltransferases are found in all kingdoms of life and produce a diverse area of glycosylated species, including bacterial natural products, peptidoglycan, glycolipids, glycoproteins, glycosaminoglycans, glycogen, and cellulose. Photoaffinity probes based on nucleotide sugars have been used to study various glycosyltransferases.
In particular, the [32P]5-azido-UDP-glucose probe (Figure 2A and described above) has been widely used to identify the nucleotide sugar binding sites of glucosyltransferases. In 1990, Frost et al. used [32P]5-azido-UDP-glucose to photolabel red beet (Beta vulgaris L.) storage tissue. They identified a 57-kDa polypeptide proposed to be the UDP-glucose-binding subunit of callose ((1,3)-β-glucan) synthase.[14] In the same year, Lin et al. used [32P]5-azido-UDP-glucose to identify the UDP-glucose-binding subunit of cellulose synthase in Acetobacter xylinum.[15] More recently, [32P]5-azido-UDP-glucose was used to identify the UDP-glucose binding region of glycogenin, the autoglucosyltransferase responsible for initiating glycogen biosynthesis in animals. Glycogenin isolated from purified rabbit skeletal muscle proteoglycogen was photolabeled with [32P]5-azido-UDP-glucose, then proteolyzed. Sequence analysis identified two labeled fragments corresponding to glycogenin's UDP-glucose-binding domain.[16]
The same [32P]5-azido-UDP-glucose probe was used to determine the membrane orientation of active sites of ER- and Golgi-resident glycosyltransferases.[17] Drake et al. performed photoaffinity labeling of microsomal preparations using the [32P]5-azido-UDPglucose reagent to determine whether glycosyltransferase active sites were in the interior or exterior of microsomal vesicles. UDP-glucose:dolichol phosphate glucosyltransferase (Glc-P-Dol synthase), an enzyme involved in the early steps of N-linked glycan biosynthesis, was labeled equally well in intact versus disrupted vesicles, indicating that the catalytic domain was located on the outside of the microsomal vesicles, corresponding to a cytoplasmic orientation in the originating ER membrane. In contrast, glucuronosyltransferases, which transfer glucuronic acid to a variety of substrates, were more efficiently labeled in disrupted vesicles, indicative of a lumenal orientation for the catalytic domains of these enzymes. Similarly, Rancour and Menon reported enzyme orientation studies using a novel photoaffinity probe based on UTP. This probe contains the 32P radiolabel at the α-phosphate and an azidoaniline photocrossinker attached to the γ-phosphate (AAUTP, Figure 2B). This reagent was used to confirm the membrane orientation of ER-resident enzymes and has the advantage that it does not include modifications to the uridine portion of the molecule, which is a key recognition element for many glycosyltransferases.[18] The AAUTP probe has also been used to selectively label and identify the UDP-GlcNAc binding subunit of the GlcNAc-phosphatidylinositol synthetic complex.[19]
Another approach to mapping glycosyltransferase active sites involved the use of a photocrosslinking reagent as a surrogate acceptor substrate, which binds the active site of the enzyme with high affinity. Xiong et al. used the novel photocrosslinker 4-azido-2-hydroxybenzoic acid (4-AzHBA, Figure 2C) to label the active site of human UDP-glucuronosyltransferase 1A10.[20] Subsequent mass spectrometry analysis of the trypsinized protein identified the crosslinked peptide (EFMVFHAQWK) and was followed by analysis of site-directed mutants to determine that two phenylalanine residues (Phe90 and Phe93) are associated with acceptor binding in this enzyme.
Energy-independent transport of hexoses, including glucose, galactose and fructose, across membranes is mediated by a family of glucose transporters (GLUTs). The GLUT family comprises five primary members, as well as additional isoforms. Over the past 25 years, the Holman group has reported a variety of photocrosslinking carbohydrate reagents, many of which have been employed in the study of GLUTs. The most advanced probes can be added to living cells, where they crosslink to extracellularly oriented GLUTs and can be used for isolation and characterization of these transporters. In early studies, this group reported bis-mannose based photoaffinity probes for labeling erythrocyte and adipocyte hexose transporters, and have shown the usefulness of radiolabeled bis-glycoside photoaffinity probes to crosslink glucose transporters in erythrocyte or adipocytes.[21,22] In 2001, the group constructed a bis-glucose probe that includes a 3-trifluoromethyl-3-phenyl-diazirine crosslinker and a biotin tag for purification (Figure 3A).[23] They incubated the bisglucose probe with rat adipocytes and irradiated the cells with UV light. After precipitating the biotinylated proteins from the lysed cells, they found the GLUT4 was isolated, although the possible existence of copurifying proteins was not investigated. The Holman group has also prepared probes selective for GLUT5, which permits transport of D-fructose. These probes incorporate 1-amino-2,5-anhydro-mannitol, a potent inhibitor of GLUT5, an aryl diazirine, and biotinylated linker (Figure 3B).[24,25] Interestingly, the affinities of the probes were significantly enhanced when there was an ethylenediamine spacer between the furanose and the biotinylated photoaffinity group. The best probe was capable of capturing GLUT5 from CHO cells expressing this protein. While all of these probes seem to be effective at capturing the intended sugar transporter targets, their selectivities have not yet been assessed.
Glycans, which may be attached to proteins or lipids, are critical mediators of macromolecular recognition events, playing key roles in biological processes such as cell adhesion or endocytosis. However, the transient nature of these interactions (low binding affinities and rapid dissociation rates) often precludes their characterization, since biologically relevant complexes may not be able to withstand traditional purification. As will be described in the following section, the utilization of photocrosslinkers that can create a covalent bond between glycoconjugates and their binding partners offers a powerful strategy to stabilize glycoconjugate complexes and thereby enable their characterization.
In the mid-1980s, Lee and coworkers reported the preparation of photoaffinity glycopeptides that were used to interrogate the binding properties of the asialoglycoprotein receptor. Asialoglycoprotein receptor is abundant in mammalian livers and named for its function as a lectin that interacts with galactose residues on desialylated glycoproteins.[26–28] The photoaffinity glycopeptides used in these studies were prepared from naturally occurring sources. In one such study, the authors isolated an asialoglycopeptide from bovine fetuin. This peptide contained a single N-linked glycan with a triantennary structure, upon which they introduced a single 6-amino-Gal residue at one of three positions, for subsequent incorporation of an aryl azide photocrosslinking functionality as well as a 125I label for detection (Figure 4A).[29] Using these asialoglycopeptide ligands, the authors were able to demonstrate that the regiochemical placement of the photocrosslinker resulted in differential labeling of the major (RHL1) and minor (RHL2/3) asialoglycoprotein receptor subunits, thus defining the geometry of the galactose residues in the binding site. In subsequent studies, the same group developed "glycoprobes" for a more facile synthesis of reagents for lectin labeling in complex biological samples.[30,31] These glycoprobes incorporated a digoxin tag between the photocrosslinking group and the variable glycan chain so that a general commercially available antibody could be used for visualization of the crosslinked species formed upon photoirradiation. Using this system, the authors were able to successfully label all 3 subunits of the asialoglycoprotein receptor from preparations of rat liver membranes.
Another important early example comes from the work of Borén and coworkers. They identified the adhesion factor BabA present on the surface of the pathogenic bacteria Helicobacter pylori through the use of a semisynthetically prepared glycoprotein with both an affinity-tag and photocrosslinker handle.[32] H. pylori is the causative agent for peptic ulcer disease. Adherence of this bacterium to the human gastric epithelium for subsequent colonization was known to be mediated by a fucosylated Lewis b (Leb) blood group antigen. Therefore, the authors conjugated the Leb glycan to human serum albumin, which was also modified with a disulfide-linked, multifunctional reagent containing a biotin purification handle and an aryl azide photocrosslinker. Thus, interactions between Leb glycans and their binding partners on the surface of bacteria brought the bacteria in proximity for efficient photocrosslinking to the biotinylated tag. Subsequent reduction of the disulfide bond liberated the albumin conjugate, freeing the now biotinylated bacterial receptor (Figure 4B). Biotinylation enabled detection of the receptor by immunoblot with peroxidase-streptavidin or purification of the protein using streptavidin-coated magnetic beads. Using N-terminal sequencing, the purified receptor was identified as BabA. This process was termed receptor activity-directed affinity tagging or ReTagging, and is an important early example of the power of photocrosslinking approaches applied in a cellular setting. Moreover, this example illustrates important challenges such as the utility of an affinity tag for effective purification and the requirement for ample sample to facilitate identification. It is also noteworthy that BabA's identity was determined by N-terminal sequencing. In the intervening years since this study, advances in mass spectrometry have enabled identification of proteins with much smaller quantities of sample, suggesting that approaches like ReTagging are worth revisiting since they may now be even more effective and easier to implement.
The design of effective photocrosslinking reagents is greatly aided by a priori knowledge of the structure of the glycan-binder/glycan pair to be studied. However, even in cases where this information is available, optimization is often essential. The process of probe optimization is demonstrated in work by the Pieters group aimed at developing glycan-based photoprobes to selectively label galectin-3 in different sample types (i. e., different cancerous states). The intended application for these probes is in the determination of galectin-3 levels in clinical tissues for diagnosis and prognosis assessment.[33] Recent work has focused on a new series of photoprobes based on a thiodigalactoside inhibitor of galectin-3.[34] In this work, the linker connecting the thiodigalactoside and the photoaffinity label was varied and the effects on binding affinity and specificity of labeling the target protein were assessed. Acetophenone or benzophenone photocrosslinking groups were connected through either an ester, amide, or triazole linkage to the C3 positions of the thiodigalactoside (Figure 5A). Probes also incorporated a terminal alkyne for fluorescent labeling with click chemistry. The compound with the best affinity and labeling efficiency for galectin-3 was identified experimentally and included a flexible acetophenone moiety and an amide linkage. However, rigidity seemed to be beneficial for maximizing labeling selectivity in the complex mixture of mammalian cell lysates, with the compound possessing the benzophenone moiety and triazole linkage exhibiting the highest specificity for galectin-3 labeling. This example illustrates the difficulties associated with predicting which attributes will lead to the desired properties, and how testing the compounds in a cellular system is the ultimate determinant.
Similarly, the best photocrosslinking functionality for a particular application may also need to be determined experimentally. The commonly used photocrosslinking groups vary in size, solubility, and reactivity.[1] The feature most important for a particular application may be difficult to predict. For example, Lindhorst and coworkers developed a series of photoreactive α-mannosides possessing either a diazirine, an aryl azide, or a benzophenone group attached to the C1 position (Figure 5B). These photoprobes generate carbenes, nitrenes, and radicals, respectively, upon UV irradiation.[35] The authors performed a comparison of their photocrosslinking efficiency by carrying out in vitro labeling experiments with a series of six amino acids (Thr, Ser, Tyr, Ile, Lys, and Arg) and three model peptides, and analyzing the products by mass spectrometry. While all photoprobes were able to form insertion products with at least one of the amino acids and peptides investigated, the diazirine was deemed the most useful for crosslinking to biomolecules in future studies. In addition to its favorable properties in terms of water solubility and time of irradiation required for activation, the preference of the diazirine for insertion into polar groups (such as amines and hydroxyl groups) is predicted to be advantageous for studies involving protein targets under physiological conditions.
Finally, the conformational properties of the glycan itself can influence crosslinking efficiencies. Sakushima and co-workers compared the behavior of photoprobes in which the attached sugar (at the reducing terminus of the glycan) was in the open chain or cyclic form (Figure 5C). A probe containing the closed ring sugar displayed improved photocrosslinking to a cognate lectin.[36] However, as with the previous example, these photocrosslinking experiments were performed in defined reaction mixtures. Examination of photocrosslinking efficiency and specificity in more complex biological samples will be required to fully assess the relative utility of the different photoprobe structures.
One intended application for carbohydrate-based photocrosslinking reagents is their use in defining functionally relevant carbohydrate binding sites on protein targets. Advances toward this goal have been reported by Lindhorst and coworkers. As described above, this group determined that photoreactive α-mannosides possessing a diazirine ring attached to C1 seemed to be the most suitable for labeling peptides (Figure 5B).[35] This photoprobe was incubated with an octapeptide from angiotensin II (H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-OH) and then UV irradiated to induce crosslinking. The researchers observed a 1:1 crosslinked product by MALDI-TOF-MS and ESI-MS. Further MS/MS studies revealed a preference for carbene insertion into the tyrosine residue, demonstrating that these reagents can be used to define preferential crosslinking sites (Figure 6).[37] In the future, such α-mannoside probes may be used for the analysis of more functionally relevant binding partners, such as the bacterial lectin FimH. For instance, it is still unknown if there is an auxiliary binding site utilized by this lectin that is important for host binding, and this photocapture/MS approach is well-suited to address that question. To this end, Lindhorst and coworkers have used MS to show that their diazirine-modified α-mannoside probe (Figure 5B) can crosslink and form a 1:1 adduct with the isolated N-terminal CRD of FimH.[37] They propose purifying the proteolytically digested fragments in analogous studies using a biotin-modified probe for analysis by MS/MS to identify critical amino acids mediating this interaction. As will be described in the next section, similar work using photocrosslinking to identify binding site residues in an isolated bacterial toxin has been performed with glycolipid probes as well.
In addition to simpler sugars, glycans, and glycopeptides described thus far, scientists have also introduced photocrosslinkers into glycolipids to search for their interaction partners. For instance, Schnaar and coworkers prepared glycolipid probes with the goal of more precisely defining the regions within Clostridial neurotoxins, botulinum and tetanus, that can target glycolipids as receptors for endocytosis. One of the novel aspects of this approach was the way that Schnaar and coworkers synthesized a ganglioside-based photocrosslinking reagent by introducing an aryl azide onto the C7 position of the terminal sialic acid of GD1b via a disulfide-containing linker (Figure 7A).[38] To do so, the authors first treated the GD1b with periodic acid under mild conditions to selectively oxidize the glycerol side chain of terminal sialic acids resulting in aldehyde formation. Secondly, this aldehyde was converted to an amine by a reductive amination reaction. Thirdly, they coupled the arylazido group to this amine by using a commercial photoaffinity intermediate sulfosuccinimidyl 2-(p-azidosalicylamido) ethyl-1,3′-dithiopropionate. Finally, this photoaffinity labeled GD1b probe was radioiodinated by sodium iodide (125I). The tetanus toxin C fragment (residues ~864–1315) was incubated with 125I-azido-GD1b and irradiated to induce crosslinking. Following reduction for cleavage of disulfide bond and proteolytic digestion, they observed that a single peptide fragment was 125I-labeled, and this labeling event was inhibited by preincubation of the toxin with the b-series ganglioside GT1b. The unlabeled polypeptide was sequenced and shown to encompass the 34-amino acid C-terminal domain of tetanus toxin (residues 1282–1315); further MALDI-MS analysis revealed a single modification site at His1293. With this information, the authors could then perform a sequence alignment of the C-termini of the 7 homologous Clostridial toxins (tetanus and botulinum types A-F) which possess different abilities to bind to GT1b and speculate on which amino acids had diverged to yield different glycolipid specificities over time.
Another example of a glycolipid photoprobe comes from the work of the Sonnino group. These researchers synthesized photoreactive derivatives of gangliosides (GM3, GM1, GD1b) to capture their protein binding partners found in cultured cells.[39] In particular, one such derivative of GM1 was designed to study molecules interacting with the hydrophilic end of the ganglioside. This photoprobe consisted of a photoreactive nitrophenylazide group attached via the terminal galactose on the sialyloligosaccharide (Figure 7B).[40] To prepare this probe, they oxidized a radiolabeled [3H]GM1 by galactose oxidase to produce an aldehyde, followed by a reductive amination that resulted in an amine derivative of [3H]GM1. Finally, this amine derivative was treated with 4-fluoro-3-nitrophenylazide to obtain a photoactivatable aryl azide conjugate, i. e. [3H]GM1-N3. Upon exogenous addition of [3H]GM1-N3 to cultured neuronal cells, a minor portion was recovered from sphingolipid-enriched membrane fractions (SEMFs) as determined by sucrose gradient ultracentrifugation, thus suggesting their insertion into the lipid bilayer in a physiologically relevant manner mimicking that of endogenous GM1. By biotin labeling of cell surface proteins followed by UV irradiation of treated cells, the SEMF obtained could be further affinity purified with streptavidin beads to enrich for those GM1-crosslinked proteins that possess exoplasmic domains. While the majority of biotin-labeled proteins recovered by this method were in the 50 to 70 kDa range, two radioactive bands at 135 and 35 kDa were also detected. The 135 kDa species was immunologically identified as the GPI-anchored adhesion protein TAG-1, a typical component of the SEMF from cerebellar neurons previously identified as a GM1 interaction partner. This exciting result suggests that targets can be labeled in complex systems with this approach.
Another approach for creating photoreactive glycoconjugates is to exploit the naturally occurring metabolic pathways of a cell. Simple sugars modified with a photoreactive crosslinker can be added to the media surrounding cultured cells. These precursors can be transformed by the cells, producing a pool of photocrosslinking nucleotide sugars that can be utilized by the cell's glycosyltransferases in the assembly of complex glycoconjugates. Due to the numerous possible glycan structures that are capable of being modified in the cell, advantages include being able to use the same simple sugar precursor to address various biological questions (i. e., to study different target biomolecules). As described below, this approach has been successfully applied to the examination of multiple sialic acid-modified glycoconjugates.
In 2005, Paulson and coworkers demonstrated the first use of metabolically incorporated photocrosslinking sugars to define glycan-mediated interactions. To determine the glycan-dependent cis ligands of CD22 in B cells, they prepared a photoreactive metabolite comprised of N-acetylneuraminic acid modified with an aryl azide at the C9 position (9-AAz-NeuAc).[41] BJAB K20, a B-cell line that is deficient in sialic acid biosynthesis, were used to monitor the efficiency of 9-AAz-NeuAc incorporation into cell-surface glycoconjugates. These cells were cultured in serum-free media containing 9-AAz-NeuAc. Flow cytometry analysis with a fluorescently labeled sialic acid-binding lectin (SNA, Sambucus nigra agglutinin) or a CD22-Fc chimera revealed that the cell-surface sialic acid levels of the K20 cells were similar to those of sialic acid producing B cells. Furthermore, deployment of the Staudinger ligation with a triphenylphosphine-biotin probe revealed cell surface display of the aryl azide (AAz) functional group. Upon UV irradiation and immunoblotting for CD22, cells supplemented with NeuAc gave the expected CD22 band at 120 kDa, while those cells cultured with 9-AAz-NeuAc yielded an additional higher molecular weight band of >250 kDa. Subsequent immunoprecipitation of CD22 and immunoblotting for probable interaction partners such as CD45, PMCA, IgM, and CD19 yielded no positive results. However, the authors were able to decipher that, in fact, a CD22 homomultimeric complex was being observed by performing a co-immunoprecipitation of lysates from cells simultaneously expressing both Flag- and myc-tagged versions of CD22. This method allowed a clear demonstration of the homomeric, glycan-mediated interaction of CD22 that masks this protein from other potential cis binding partners.
In more recent work, these authors combined the same photoreactive reagent with a quantitative proteomics mass spectrometry approach, enabling identification of the trans ligands of CD22 in homotypic B-cell interactions.[42] As often encountered in mass spectrometry analysis of crosslinked samples,[43] a primary challenge is to obtain a suitable quantity of crosslinked material for mass spectrometry analysis. To circumvent this challenge, the authors first generated a candidate list of interaction partners, by crosslinking a CD22-Fc chimera to 9-AAz-NeuAc-labeled B cells (K20) and immunoaffinity-capturing the complexes. The higher molecular weight crosslinked complexes were then subjected to in-gel trypsin digestion and nano-LC-MS/MS, whereby (based on the spectral count values compared with that of non-irradiated lysates) 24 candidate proteins were identified, 18 of which were known glycoproteins and carried on for further analysis. In an alternative approach, the authors performed similar experiments under SILAC conditions with stable isotopes of lysine and arginine. Thus, differentially labeled heavy or light 9-AAz-NeuAc-labeled B cells (K20) were both incubated with the CD22-Fc chimera, but only one set was subjected to UV irradiation. The cell lysates were subsequently combined and purified by immunoaffinity capture. The samples were then subjected to on-bead trypsin digestion and MuDPITMS/MS, whereby 19 candidate proteins were identified, all of which were membrane glycoproteins. Taken together, these two proteomics approaches yielded a total 27 candidate glycoproteins (10 candidates were identified by both approaches). In order to determine which of these candidates are bone fide trans ligands for CD22 in the context of contacting cells, 9-AAz-NeuAc-labeled B cells (K20) were overlaid on a CHO cell monolayer expressing CD22 with a C-terminal V5 tag and UV irradiated to induce photocrosslinking (Figure 8). Anti-V5 immunoprecipitates were subjected to immunoblot analysis with antibodies for 11 of the 27 candidate proteins, and of these only IgM, CD45, and Basigin appeared in crosslinked complexes. Further support for these findings came from the observation that redistribution of the most robust ligand, IgM, to sites of contacting B cells was decreased in mice lacking CD22 or the sialyltransferase responsible for its sialylation. This work illustrates challenges associated with characterizing photocrosslinked complexes and the importance of developing methods to verify that interaction partners observed in vitro are biologically relevant.
The Kohler group also captured CD22 homooligomerization in B cells, this time utilizing a diazirine photocrosslinker located on the N-acyl side chain of sialic acid (SiaDAz). The diazirine can be incorporated into glycoconjugates by culturing cells with a protected form of SiaDAz, or its mannosamine precursor (ManNDAz) (Figure 9).[44] SiaDAz has been demonstrated to effectively compete with endogenous sialic acid for incorporation into glycoconjugates in several cell types, including B cells (BJAB K88) and T cells (Jurkat).[44,45] The Kohler group has gone on to use SiaDAz to crosslink ganglioside-protein interactions.[46] First, they used MALDI-TOF-MS to confirm that Jurkat T cells cultured with Ac4ManNDAz produce SiaDAz-modified glycolipids, including GM1 and GM2. In addition, GM1-SiaDAz extracted from these cells could still be recognized by a known binding partner, cholera toxin subunit B (CTxB). Furthermore, when Jurkat T cells were cultured with Ac4ManNDAz, incubated with CTxB, and UV-irradiated, a new toxin-containing species was formed, which was identified by SAMDI-TOF-MS to be covalently crosslinked CTxB-GM1-SiaDAz.[45] This group is currently pursing use of the SiaDAz probe, in concert with mass spectrometry approaches, to identify novel sialoside-mediated binding interactions. In addition, efforts are underway to introduce photocrosslinkers into classes of glycans other than sialosides.[47]
The incorporation of a photocrosslinker on a carbohydrate unavoidably results in a structural change that can potentially alter recognition events. Therefore, the identity of the photocrosslinking group and its position of attachment onto the sugar are key aspects of experimental design. In the case of sialic acid, modifications are known to have a substantial impact on glycan recognition. An important concern is that modifications might sterically interfere with recognition events, so the use of smaller photocrosslinking moieties (diazirine rather than a benzophenone) may be advised. In particular, modifications to the O9 or Na-acyl positions of sialosides have been shown to result in significant alterations in the affinity and selectivity of recognition by sialoside binding proteins, such as sialoadhesin, myelin associated glycoprotein, and CD22.[48] Surprisingly, in some cases, aromatic modification of sialosides at the C9 position of sialic acid can significantly increase binding affinity.[49] Indeed, the 9-AAz-NeuAc reagent described above [41] likely enhances CD22 recognition events.
Examination of structural data for sialoside recognition complexes reveals that the C9-modified 9-AAz-NeuAc and the C5-modified SiaDAz are likely to have complementary applications. For example, the crystal structures of murine sialoadhesin show that tryptophan 2 is in van der Waals contact with the acetamido methyl group, likely hindering recognition of sialosides modified on sialic acid's N-acyl position.[50,51] Indeed, sialoadhesin preferentially binds NeuAc, over both natural and unnatural N-acyl variants.[52,53] On the other hand, a small hydrophobic pocket enables sialoadhesin to recognize sialosides with aromatic modifications at the C9 position of sialic acid. Thus, the 9-AAz-NeuAc is likely the preferred reagent to examine sialoadhesin binding partners. The crystal structure of cholera toxin complexes with the GM1 glycan shows a somewhat contrasting picture.[54] In this complex, the N-acyl position of sialic acid is highly accessible, while O9 engages in a hydrogen bound with glutamine 56. In this case, SiaDAz may be the preferred reagent to capture the binding interaction.
In addition, in nature, sialic acids are subject to a wide variety of post-glycosylational modifications that can dramatically alter their binding properties and functions.[55] Introduction of photocrosslinking groups may prevent these modifications and interfere with study of interactions that require variant forms of sialic acid. Again, the 9-AAz-NeuAc and SiaDAz reagents offer complementary uses. C9-modified compounds can be used to examine interactions in which N-glycolyl or deaminated sialic acids are required. N-acyl-modifed compounds can be used in situations where O9-acetylation, - lactylation, or –phosphorylation is physiologically important.
For the discovery of unknown binding events in physiological samples, which is likely the most important application for these approaches, data describing sialic acid recognition and modifications will often be unavailable or incomplete. In these cases, rational selection of the preferred photocrosslinking reagent is impossible. Therefore, the best strategy will likely be to empirically determine the best photocrosslinking reagent for a particular application, using the panel of reagents that have been shown to have at least reasonably efficient metabolic incorporation.
As described above, the current arsenal of carbohydrate photoprobes comprises a diverse class of reagents. The carbohydrate portions of these reagents have been based on naturally occurring glycoconjugates, synthetic glycans, and carbohydrate analogs. These targeting motifs enable binding to all classes of carbohydrate-binding proteins and metabolic enzymes. Biocompatible photocrosslinking groups enable selective crosslinking, even in complex lysates or cellular settings. Finally, the use of affinity purification tags allows isolation of crosslinked complexes for further characterization. While early work in this area demonstrated effective crosslinking of carbohydrate photoprobes, characterization of the crosslinked complexes remained a challenge. However, new developments in analytical methods are providing solutions to this difficulty. Recently, the use of Huisgen 1,3-dipolar cycloaddition chemistry has enabled the deployment of small functional groups (alkynes and azides) as handles for subsequent attachment of affinity purification tags. This strategy allows for effective purification of crosslinked species from complex mixtures. In addition, new mass spectrometry methods facilitate identification of crosslinked molecules and are beginning to allow delineation of crosslinking sites. Taken together, the use of carbohydrate photoprobes, in combination with modern analytical methods, is poised to provide new essential new insights into the biological roles of carbohydrates.
We acknowledge support from the NIH (R01GM090271 and R21AI094427) and from the Welch Foundation (I-1686). J. J. K. is an Alfred P. Sloan Research Fellow.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.

subset: commoncrawl


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- Product Dimensions: 5.8 x 4.1 x 13.5 cm ; 172 g
- Shipping Weight: 1.3 Kg
- Item model number: ES-WD51-P
- Date first available at Amazon.ca: Oct. 20 2010
- Average Customer Review: 41 customer reviews
- Amazon Bestsellers Rank: #6,525 in Health & Personal Care (See Top 100 in Health & Personal Care)
Panasonic ES-WD51-P Epiglide Ladies Wet Dry Epilator (Pink)
- Fully immersible, the epilator can be used in the bath with lather for a refreshing, regenerating experience, or used dry wherever, whenever it's convenient.
- 48 precise tweezers capture long and fine hairs easily
- The pivoting head's side-to-side gliding motion treats skin softly, gently removing hair from the root.
- 3-in-1 hair removal system, shaver and bikini trimmer also included, giving you an all-in-one hair-removal package. The Bikini attachment and skin protector is pre-assembled on the shaver in the packaging
- Nickel-free, hypo-allergenic blades for sensitive skin types
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The product is easy to use, it looks exactly like the photo, maybe a little smaller.
I am satisfied with my purchase and would definitely recommend this product to a friend!
Pls note- I only tried wet on one occasion and found it uneffective on my texture of hair and never did again, so my review is based on dry epilating only after a warm shower and on legs only...
Most recent customer reviews
I purchased this epilator back in Aug 2013 and I'm happy to say that it's still working well. I use it at least a couple times a week, usually dry though I have used it wet a few... Read morePublished 3 months ago by Leanne C
My very first epilator was Braun. So maybe I was spoiled by its painless and easy usability. This was very disappointing when compared where that but it's fine for the price. Read morePublished 4 months ago by Ally
Really great epilator with a nice battery life in the beginning. I've had it for over two years and have not experienced any product defects or difficulties. Read morePublished 7 months ago by Shopaholica
I love my Epilady. I used to make homemade body sugar, but it was hit-and-miss and truly a chore. This gadget achieves the same effect with none of the hassle. Read morePublished 10 months ago by PJF
I have had the Discrette Epilady. It is faster and far more effective. It also has 2 speeds. If I could return the ES WD51-P, I would.
It is very slow, one speed only. Read more
Really great product. The only thing I would change is it gets very slippery when wet. If your grip moves even slightly you end up knocking the button on the side that releases the... Read morePublished 12 months ago by virginia adeney
I wanted to like this. Hardly pulled any hair out at all but it sure did hurt a lot. The shaver attachment doesn't work well either not shaving at all.Published 15 months ago by Nikki
This thing is great - and it comes with a charger which is awesome.Published 19 months ago by Bina Chauhan


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The War Against Paris, 1871by Robert Tombs
Pub. Date: 12/03/1981
Publisher: Cambridge University Press
The Paris Commune of 1871 is one of the great romantic failures in revolutionary history. Yet very little is known about its enemies, and especially the army, which first fraternized with the revolutionaries and then, two months later, crushed them with the utmost violence. This book, based on extensive archival research, is the first serious study of the role of the army in the civil war. It examines its composition and organization, its weaknesses and their effect on government policy, the steps taken to improve morale and discipline, the state of mind of officers and men and, finally, the conduct of the army in battle and the causes of the final bloodshed, in which about 20,000 Parisians were killed in the fighting or executed afterwards. Its purpose is to cast new light on the policy of the government and the problems of using an army in a civil war, and to tell for the first time the full tragedy of the suppression of the Comune, one of the bloodiest and least understood social conflicts in the history of modern Europe.
Product Details
- 9780521287845
- Publisher:
- Cambridge University Press
- Publication date:
- 12/03/1981
- Edition description:
- New Edition
- Pages:
- 256
- Product dimensions:
- 5.98(w) x 8.98(h) x 0.63(d)
Table of ContentsList of illustrations; Preface; Acknowledgements; Glossary; 1. Wars and rumours of wars; 2. A defeated army; 3. Insurrection;4. Interlude; 5. Civil war; 6. The Army of Versailles; 7. Propaganda, myth and the army; 8. The second siege; 9. The battle of Paris; 10. Expiation; 11. Conclusion; Appendices; Notes; Bibliography; Index.
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Military Wedding - By mary
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They take the fear and disbelief of the sheep of this Country and turn it into a driving force of anger and vengeance towards law abiding gun owners.
Gun Banners
Grasping at Straws in the North Woods
The NRA understands the proposed law all too clearly. As for the MRGO, focusing on the origin of photographs as evidence of intentional distortion is pretty feeble:
Shannon Watts Blames NRA for Disarming of Courthouse Security Officer
Turns out gun banning, freedom hater, Shannon Watts, should have asked a few more questions before, once again, publicly humiliating herself.
Pseudoscience About Guns in the Canadian Medical Association Journal
Gary Mauser, PhD's, analysis of a "study" claiming that Australia's 1996 gun confiscation law ended "fatal mass shootings" there. Turns out it didn't…
Ten Favorite Lies of the Gun Control Lobby
One can only conclude that the gun control lobby liars are more interested in political posturing and cultural warfare than in reducing "gun violence."
The AMA, Ethics and Gun Control (Part II)
Political expediency and social embarrassment, about kinky sex and lies, perjury, and the appearance of JAMA and the AMA closing ranks behind a disgraced president…
Another California Anti-Gun Legislator in Trouble with the Law
After two agonizing months, the ex-wife of gun grabbing Democratic Assemblyman Roger Hernández finally received her restraining order against the alleged domestic abuser from Los Angeles County..
Gun Banners Schedule Recruitment Rally In Naperville, IL
The gun control movement has scheduled an antigun propaganda film viewing and gun control recruitment rally in the western suburbs of Illinois…
The Truth Behind the Denying Firearms & Explosives to Dangerous Terrorists Act
The worst part of this bill is that it removes a fundamental right of every United States citizen – due process.
Another Gun Research Peer Review FAIL at JAMA
The editors at one of America's leading medical journals have been clear for years on what they want readers to think about guns—private ownership of firearms should be banned..
Are We Just like Venezuela?
Democrats call strong-arm gun-confiscation "common-sense regulation," so we can become a "gun-free utopia," just like Venezuela!
Anti-Gun Senator Ayotte Still Needs To Hear From You
It seems that Senator Ayotte has forgotten what she learned in law school, that in America, people are innocent until proven guilty…
American Medical Association, Ethics & Gun Control (Part I)
I can testify that the AMA has been moving steadily to the left in a variety of issues, from the perversion of medical ethics to gun control…
Organized Medicine's Answer To The Orlando Mass Murder: Deception & Control
True to form, the American Academy of Pediatrics beats the dead horse of "gun violence" in its statement responding to Sunday's mass murder in Orlando…
Hillary's Position Against Guns in Her Own Words
One thing is clear; Hillary is in fact coming for your guns. She does not respect your right to own a gun, does not believe that right is protected by the constitution and is willing to flat out lie..
Gun Control History and the NRA
History may not repeat itself but it often rhymes, and gun grabbing politicians are driven by politics, not saving lives. Until that stops nothing will change…
Real Public Health Gun Experts Debunk 'The Trace'
A rabidly anti-gun website is helpfully coaching us on how we can make guns safer for everyone. If you believe that, your probably gonna voter for Hillary too…
LA: 2016 Legislative Session Ends, Gun Rights still at Risk in New Orleans
On April 22, 2016, anti-gun Mayor of New Orleans, Mitch Landrieu, unveiled a new gun control initiative that he planned to impose upon the City of New Orleans..
'Under the Gun' Director, Stephanie Soechtig, Confesses to Federal Gun Crimes
Ms. Stephanie Soechtig excitedly states in a video interview with The Lip TV how she brazenly broke federal gun laws for Katie Couric's anti gun hit piece Under the Gun..
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Are we seeing the last throws of the anti-gun left in CA. As they willing eat their own…
Always Trying To Find A Way To Strangle Your Second Amendment Rights
Ways they try to do this is to cut off supply, drive prices up, make gun ownership inconvenient, if not impossible, and attempt to make gun owners feel isolated and even threatened…
Unbelievably: Katie Couric "Apologizes" with Second Misleading Edit, Doubling Down
On the obscure web page where Katie buries her apology. The problem is that the transcript stops only about one-quarter of the way into the response, clipping off the most relevant parts of the answer
Two Profiles in Hoplophobia
At Doctors for Responsible Gun Ownership we will relentlessly expose anti-gun rights extremists using their lofty university positions to attack our civil rights.
Elites Double Down on Medical Gun Control Politics
Medical gun control activists are receiving pushback from sick & injured people who come to them for help but instead find pressure to give up their civil right to own a gun.
Doctors Kill 23 Times More People Than Guns Kill
The horrifying number that doctors & hospitals kill a quarter million Americans every year, certainly far exceeds the number killed by guns..,
Donald Trump: 'Hillary Clinton Wants to Take Your Guns'
Pressing the point, Trump declared, "Hillary Clinton wants to take your guns away and she wants to abolish the Second Amendment."
The Dogs of War Are Close ~ American Spring
I will state here & now, for the record, if these demented individuals succeed in passing laws to restrict, ban or confiscate firearms, THERE WILL BE BLOOD.
New Jersey: Senate Committee Passes Anti-Gun Legislation on Party-Line Vote
Undeterred by the NJ's failed, draconian gun laws, Senator Majority Leader Loretta Weinberg (D-37) has quickly schemed to block Governor Chris Christie's pro gun executive action.
Anti-Gunner's Glossary of Words You Are Using Wrong
It's become clear over the past 30 years that, in the field of demonizing firearms and those who use them, there are equally contradictory uses of vocabulary.
You Won't Believe This, CA Politicians Hijack Bills, Insert Gun Control
Senate and Assembly Democrats "gutted and amended" 4 bills that were stalled and changed them into 4 brand new, ANTI-GUN measures.
Obama Seeks To Restrict 2nd Amendment Rights For Seniors
Any senior whose funds, including Social Security Benefits, are managed by someone else, could trigger the SSA alerting authorities to revoke your permit to own and/or carry a gun…
Aging Rocker to College Kids: You're Too Drunk, Violent, & Stupid for Your Rights
It's an incredibly condescending outlook that would demand college students meekly submit to whatever victimization a predator might choose for them.
Hillary's "Australian-Style" Solution
It's looking very likely that Hillary Clinton will avoid going to prison for her illegal handling of classified emails, so we need to consider what a Clinton presidency means to gun owners…
Gun Banner Weinberg Pushing Mandatory Mental-Health Evaluations for NJ Gun Owner
Gun Banning Senator Loretta Weinberg is pushing mandatory mental-health evaluations if you own or want to purchase a firearm in NJ…
Gabby Gifford & Gun Grabber Husband Target Illinois
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Gabby Giffords & 100,000's of Americans Are Gun Criminals by Definition
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Hoplophobic Doctors push for More Bogus Gun Research at Taxpayer's Expense
Sitting through last week's GunMeggedon in the California Senate's Public Safety Committee was enough to raise one's blood pressure to dangerous levels.
NASCAR Affiliated Lobbyist Now Working to Ban Fan's Guns in New Hampshire
The same lobbyists who are employed by the NH International Motor Speedway & NASCAR are now working to ban your guns in New Hampshire…
Hillary Clinton's Ignorant Stance on Gun Violence & Gun Manufacturers
Who cares about the facts? When her gun control agenda is focused on emotion, regardless of the truth…
DRGO's 1996 Congressional Testimony: Defunding Gun Control Politics at the CDC
In the 1990s influential bureaucrats at the CDC found another deadly "disease," gun violence…


text:
Lesbian Dating in Benton
USA > Arkansas > Benton Lesbians
Love, Adventure, Nature, Fun.
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Harry Potter And The Prisoner Of Azkaban, Film Score: The Werewolf Scene
Features of This Tracka strongly dramatic aesthetic
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Rick Fox, Kurt Warner in 'DWTS'
Retired NBA player Rick Fox thinks it's not fair that people expect him to do well in the 11th season of ABC's "Dancing with the Stars," which begins Monday night.
Yes, athletes have done well in the past. NFL Hall of Famer Emmitt Smith, IndyCar driver Helio Castroneves and Olympic gold medalists Apolo Anton Ohno, Kristi Yamaguchi and Shawn Johnson all have won.
But Fox would prefer to be compared to the other athlete -- future NFL Hall of Famer Kurt Warner -- in this fall's edition of the top-five-rated show.
"All the pressure should be on Kurt. The football players have represented themselves well on this show," said Fox, who has been acting since retiring from the NBA in 2004. "But basketball? Come on. Did you see Clyde Drexler dance in 2007? Ugh."
The real favorites will be Jennifer Grey, who rose to fame starring in "Dirty Dancing," and singer Brandy, who has done a lot of dancing in her music videos.
Brandy is downplaying being considered one of the favorites.
"Dancing is never one of my strongest points. I'm pigeon-toed naturally," she said. "My posture is off. I'm really nervous. All this stuff is new to me."
Fox laughed at Brandy, saying: "I'm the one pigeon-toed. You should be voting for me. I have one week to prove to the world I can dance. Help me!"
Here's all the info you'll ever need on this season's edition of "Dancing With The Stars."
Here's a look at this fall's competitors:
Michael Bolton: The love-song guru of the 1990s didn't do much dancing in his music videos, save some tame back-and-forth swaying. Bolton, 57, will be dancing with Chelsie Hightower, the 21-year-old who, through three seasons on the show, has never danced with a top-three competitor.
Brandy: Early reports out of the "DWTS" camp indicate that the singer-actress is one of the favorites; her athletic build and competitive persona back that up. Partner Maksim Chmerkovskiy has a history of dealing well with fierce types, and he most recently helped ESPN's Erin Andrews to a third-place finish in Season 10.
Margaret Cho: The burlesque-dancing comedian will be paired with Louis van Amstel, who opened his "DWTS" career with a last-place finish but has since rebounded, most recently dancing with third-place Kelly Osbourne in the ninth season.
Jennifer Grey: The odds-on favorite to take the mirror-ball trophy, Grey's experience in "Dirty Dancing" should carry her far in the competition. The fact that she's dancing with Derek Hough -- who has danced with two winners (Brooke Burke in Season 7 and Nicole Scherzinger in Season 10) in his five stints on the show -- shouldn't hurt.
Rick Fox: Partner Cheryl Burke has routinely been paired with sports stars, including dancing with Season 3 winner Emmitt Smith and Chad Ochocinco in Season 10, and the agile Fox has the body type to excel -- much like winners Shawn Johnson (Season 8), Kristi Yamaguchi (Season 6) and Apolo Anton Ohno (Season 4) did.
David Hasselhoff: Hasselhoff is still enjoying the fame bump from his Comedy Central roast and is paired with Kym Johnson, who has a history of succeeding with former television icons, including a victory with Donny Osmond in Season 9 and a Season 3 stint with Jerry Springer.
Florence Henderson: For the 76-year-old Henderson, the oldest contestant this season, only Corky Ballas, who danced with the 84-year-old Cloris Leachman in Season 7, will do.
Kyle Massey: On the other end of the age spectrum is Massey, who at 19 is this season's youngest competitor. The Disney Channel child star will dance with Lacey Schwimmer, the 22-year-old spark plug who danced with Season 7 bronze medalist Lance Bass.
Bristol Palin: Palin's partner, Mark Ballas, is a two-time champion on "DWTS" and has a history of success with younger dancers. Chmerkovskiy, however, recently said in an interview with E! Online that Palin was "a little nervous" and "a little bit out of place."
Audrina Patridge: Tony Dovolani has been pigeonholed as the resident reality-star partner on the show; he just completed a Season 10 stint with Kate Gosselin and has Melissa Rycroft on his résumé.
Mike "The Situation" Sorrentino: Speaking of reality stars, The Situation will be dancing with Karina Smirnoff, the Ukranian beauty who has finished in the top three only once in seven seasons.
Kurt Warner: The former Super Bowl champion and NFL MVP was never the most agile quarterback -- he finished his 12-year career with only 286 rushing yards -- but he'll have to move in order to keep up with Anna Trebunskaya, who finished in third place with Jerry Rice and Evan Lysacek.
The action begins Monday night, and Fox has a plan if he trips or falls.
"Wait, what do you mean the show is live? We don't do retakes?" Fox asked. "Well, I'm going to treat it like a basketball game. I'll just blame someone else."


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Related quotes
"Pain , happiness, peace, love, friends and your partner, is very common ! Every body does have these facts! But remember the partner who made you cry, you are not the only person who crying for a men ! There are many more women's who crying every single minute for a men in this planet including Insects to Animals! End of the day we all have a thing called HEART!"
"My heart is feeling up with pain. Feeling so sad and depressed wishing that my love for you can fade away.. Just seeing you with her saying I love you more than anything. after a week of dating her.. Why do I have to feel this pain?"
"I know quickly whether a guy is boyfriend material. If I can have a good time doing absolutely nothing with him, then that's boyfriend material for me. Like if we're able to have fun at a gas station. I've had some really good times at gas stations."


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Women's Health
Life With an Autoimmune Disease
Your first symptoms of an autoimmune disease may be general, such as fatigue, low-grade fever, and difficulty concentrating, making autoimmune diseases difficult to diagnose at first. You also may feel depressed and consult a doctor for that.
According to Mary J. Shomon, author of the book Living Well With Autoimmune Disease: What Your Doctor Doesn't Tell You ... That You Need to Know, what ensues after registering these complaints may be an odyssey to pinpoint which of the almost 60 different autoimmune disorders you might have, all of which affect the body differently.
Recommended Related to Women
By Meryl Davids Landau When you were in your 20s and 30s, you probably ignored random aches or other minor physical annoyances, and they usually went away. But now those symptoms can come back — often with a different cause, and calling for more serious attention.
Read the Old Symptoms, New Risks article > >
About 50 million Americans -- the vast majority of them women, especially women of working and childbearing age -- suffer from autoimmune ailments. Rheumatoid arthritis, type I diabetes, psoriasis, alopecia, lupus, thyroid disease, Addison's disease, pernicious anemia, celiac disease, multiple sclerosis, myasthenia gravis, Guillain-Barre syndrome -- these are just a few of the ailments that scientists now think stem from a common phenomenon: the activation of the body's immune system against the body itself. Also suspected of having this as a component are chronic fatigue syndrome and fibromyalgia.
Traits in Common
That such different-seeming diseases as psoriasis and diabetes could stem from a common cause actually is a relatively new notion, according to Noel R. Rose, MD, PhD, professor of molecular microbiology and immunology and pathology at the Johns Hopkins University in Baltimore. Back in the early days of the last century, he says, the idea took hold that if the immune system were to benefit us, it would have to be warding off foreign invaders from outside the body.
Now, scientists know that the immune system is a set of actions and reactions that can be triggered by a number of things besides an invading germ, virus, or bacteria. One thing that puts you at risk for being attacked by your own immune system is your genetics, says Rose. In other words, if your parents have a predisposition to autoimmune disease, you may, too. "And it's an overlapping inheritance," Rose says. "If you have one autoimmune disease, you may have more -- and you may have different ones than your parent did (or your siblings do)."
Today on WebMD
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Welcome to Fenway Health's Behavioral Health Department
There are many resources available if you or a friend or loved one is struggling with addiction to alcohol or drugs.
Fenway's Behavioral Health Department offers counseling and support groups designed to help with addiction issues, especially for people who are lesbian, gay, bisexual and transgender.
In addition, Fenway's acupuncture detox program offers walk-in sessions available on a sliding fee scale to anyone who is concerned about their substance use, with most participants paying very little or nothing. Sessions are Monday through Friday from 8:00 am to 10:00 am and 4:30 pm to 6:30 pm, and from 9:00 am to 11:00 am on Saturday mornings. Pre-registration is required. Please call 617.927.6202 to schedule your enrollment visit.
A full list of substance abuse services is shown below. You must contact Denise at 617.927.6202 for an intake appointment before accessing these services.
- Acupuncture daily walk-in clinic for alcohol and drug detox
Monday-Friday, 8:00 am – 10:00 am (last treatment at 9:15 am)
Monday-Friday, 4:30 pm – 6:30 pm(last treatment at 5:45 pm)
Saturday, 9:00 am – 11:00 am (last treatment at 10:15 am)
- Substance abuse counseling, education and case management
- Individual and group counseling
- Smoking cessation
- Relapse prevention
- HIV risk reduction education and referrals to HIV testing
- Self-Help meetings on-site


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In Android, I am writing an app where the user enters text into an
EditText view, and I would like to intercept that text before the system actually inserts it into the view, so that I can first wrap it in a
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Is there any way to do this, or do I need to wait until the text is inserted, and then replace it in the
afterTextChanged method (which seems like a waste of processing!)
Any ideas?


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The Desert Vista softball team advanced to the second round of the Division I state tournament with a 4-1 win on Saturday over No. 16 Highland.
The 17th-seeded Thunder travel to No. 1 Gilbert on Tuesday.
The Mountain Pointe softball team, seeded 12th, lost 7-4 Saturday to No. 21 North Canyon.
The 10th-seeded Desert Vista baseball team was eliminated by No. 23 Valley Vista when the Monsoon scored seven times in the first inning to set the tone for the 12-7 loss for the Thunder in its DI state playoff game.
The Pride baseball team, seeded sixth, earned a first-round bye and hosts No. 22 Gilbert on Tuesday.


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Developing Events:
- Agenda
- Banquet
- Guest Speaker (Bob Goff)
- Council in Action
- Luncheons and Breakfast
- Childcare Registration
- Worship
- Mission Congregation Dinner
You're Invited – from St. Alban's Episcopal Church, Harlingen!
For a complete summary of our three days at Council 2016, look for a four-page insert in the January/February 2016 issue of the Church News. You're Invited to Council 2016 in McAllen!
Check back often for more information. Or signup for the Council 2016 blog "Entries" RSS feed and be notified when changes are made to this website.


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Shelford House
Shelford House fits among Wine Country splendor with its Victorian elegance from 1885. The house itself is surrounded by a vast vineyard between Healdsburg and the Mendocino Coast; and if you're a fan of the Queen Anne Victorian style, it's just for you. A spacious Carriage House complements three stunning rooms in the main house, complete with... more
Golden Eagle Vacation Rentals
Golden Eagle Vacation Rentals is the perfect 'romantic getaway' and the perfect venue to launch daily excursions. Trinidad is designated as a California Coastal National Monument Gateway. Enjoy comfortable accommodations featuring wall-hung flat screen televisions with cable, full kitchens with microwaves, refrigerators and granite counter tops,... more
Sanford House B & B Inn
From start to finish, your getaway will be enhanced by the elegance and warmth of the Sanford House. The family that maintains this wonderful place has put a little of themselves into every room, as much with the antiques as in the modern amenities including private baths. A large front lawn complements the garden, with benches, koi pond and cherry... more
Annie's Jughandle Inn
Take some time out an Annie's informal and relaxed Jughandle Beach Inn in its romantic country setting four miles above the quaint village of Mendocino, California and three miles below the Noyo Harbor of Fort Bragg. The guestrooms all feature private baths, comfortable quilt-covered queen sized beds, fresh flowers and Pacific ocean breezes.... more
Antioch Ranch Christian Retreat
Redwoods, orchards and rolling hills accentuate the Antioch Ranch Christian Retreat near Mendocino. These 20 beautiful acres provide a combination of wooded seclusion and stunning natural beauty, with the unforgettable air of the California coast just nearby. These comfortable cottages each have their own ambiance, brought to life with... more
Glass Beach Inn
The Glass Beach Inn is a beautiful 1920s home surrounded by fun and adventure. This is the perfect place to unwind, be charmed and enjoy days of whale watching and riding the historic Skunk Train; or, take a refreshing stroll through the nearby Botanical Gardens. After a fascinating day, rest in any of nine elegant room with private baths; there's... more
Whether you'd prefer a small oceanview cottage or a larger vacation home, you'll find a fully furnished home away from home at Cottages at Little River Grove. Enjoy amenities like a full kitchen with high quality kitchenware, cozy gas fireplaces, and cable televisions. You will also enjoy grocery shopping and delivery service, so you won't have to... more
Jenner Inn and Cottages
The Jenner Inn & Cottages is a romantic escape where the Russian River meets the Pacific Ocean. Immerse yourself in the Sonoma coast's mighty redwoods, beautiful beaches and countless wineries – all a mere 90 minutes from San Francisco! Choose from creekside and waterfront rooms, or cottages and vacation homes, each decorated with antiques... more
Get away from it all at Ravenridge Cottages. Enter a private gate, follow a gravel road through vineyard and forest, and arrive at two comfortable cottages nestled beneath an ancient live oak. Inside, you will find full kitchens, soft beds, and cozy wood burning stoves. You will enjoy a dramatic view of dramatic hillside vistas from your... more
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A conscious nod to architect Felix Candela, this 2005 project is set to be "the newest jewel in Valencia's crown." Not only that, it's a superbly expressive use of thin shell concrete hypars.
RELATED: Architecture
Friday, 16 March 2007
City of Arts & Oceanographic Sciences - Santiago Calatrava
2 comments:
- Anonymous16 Mar 2007, 05:18:00
I like the design a lot, even if it is a bit 'opera house'. That rock pool area will look old very quickly though. It is a shame they did not do more with the landscaping.ReplyDelete
oh my! What a beautiful bit of engineering.ReplyDelete
Say what you mean, and mean what you say.
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View assessor property records for Cicero IL. The median sales price for homes in Cicero IL was $70,000. The average list price for homes for sale in Cicero on Trulia is $101,710. The median sales price decreased 22.2% compared to the prior year based on 130 sales, which increased 14.0% compared to the prior year.


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But lost in the tidal wave of media and political/civic/business-honcho gush over the May 29 groundbreaking for Marcus' Georgia Aquarium, is a nagging little question: Is this really how we build a great city?
Just a short couple of miles from the aquarium's site near Centennial Olympic Park, you can find another vision for Atlanta. On the moonscape rubble of an old concrete recycling plant in Ormewood Park, a $100-million experiment in what's dubbed New Urbanism is under way. It's the dream of Charles Brewer, who founded and left Mindspring (now EarthLink) and who, like Marcus, needed to exercise all of those under-worked dollars in his bank account.
Let's have some fun and explore the two concepts: the Atlanta Marcus wants, and the city Brewer dreams about.
At the heart of what Marcus preaches is turning Atlanta into a tourist destination. "People will want to come here from other states," he effusively told the adoring crowd at the groundbreaking. "New York has Broadway. San Francisco has the waterfront."
And somehow, Bernie's Fish Tank is going to be competition for the Big Apple and Frisco. Right.
To draw the crowds Marcus is talking about, you'd need a much bigger critical mass of attractions than an aquarium and its companion, a building full of Coca-Cola puffery. Little Deepo can't compete with the roaring Mouse just a few hours south of here. Not to mention that aquariums are seldom high-repeat-visit destinations. And the more educational the fishbowls become, the more droves of people avoid them.
About the best you can say of Marcus' logic is that he might not have thought this concept through. Or maybe it's irrelevant. My read is that he wants a latter-day pyramid glorifying Bernie Marcus.
So, if you take Marcus' real driving motivation -- mega-self-aggrandizement disguised as a civic contribution -- and heavily season it with Atlanta's trademark gullibility, voila!, we have an aquarium.
For decades, cities have wrestled with how to revitalize downtowns. No doubt things are currently happening in Atlanta's downtown -- without, it should be noted, the aid of a theme park. I won't go so far as suggesting that to reanimate many other parts of Atlanta's core would be equivalent to getting a week-old corpse at the morgue to do the Macarena. But it's close.
At least if you do it Marcus' way, which I'll call the "Big Bang." It goes like this: Hucksters periodically hit the roads for cities with decayed downtowns. In their salesmen's suitcases is the Big Bang du jour . One year, their bag of tricks will be hugely expensive heavy-rail systems -- MARTA. The next year it may be a "festival marketplace," such as Boston's Faneuil Hall, which worked - but most of its clones didn't (see Underground Atlanta). Performing arts centers? The same.
Some cities -- Denver comes to mind -- heavily invested in entire historic districts, turning old warehouses and storefronts into entertainment meccas, often anchored by big sports or cultural facilities. Call them Bigger Bang cities. They, too, worked for a while, but most also left the burgs with just another layer of urban failure.
Then, there are aquariums. I was rather surprised at Marcus' choice of monuments. After all, Home Depot was a novel concept. But aquariums? The nation is already awash in them. Many have failed - Long Beach, Calif., Denver and Tampa were colossal disasters. And, in general, as veteran Maui Ocean Center manager John Tighe commented in February: "Unfortunately, there is a tendency to build aquariums much larger and grandiose than the business model will support."
Key to that fatal optimism is overestimating attendance and underestimating operating costs. In Tampa, "consultants" (the operative syllable is "con") said the Florida Aquarium would draw about 2 million people a year (exactly what Marcus says the Georgia version will attract). About 1 million of those people never showed up the first year in Tampa, and numbers plummeted from there. The debt-strapped "privately funded" facility cost Tampa a $104 million taxpayer bailout.
Admittedly, Marcus' aquarium has a big advantage: His $200 million contribution will ensure that there's no mortgage payment. So the aquarium will probably succeed -- or die a very, very slow death. He's been thin on details about exhibits, ticket prices and the like -- so it's hard to augur the aquarium's chances.
There is a similarity with Tampa, however. Both were envisioned as reinvigorating downtown areas. It didn't happen in Tampa. That city's Channelside district is still the pits.