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Glycobiology and Extracellular Matrices| Volume 284, ISSUE 10, P6311-6321, March 06, 2009
The Binding Properties of Minimal Oligosaccharides Reveal a Common Heparan Sulfate/Dermatan Sulfate-binding Site in Hepatocyte Growth Factor/Scatter Factor That Can Accommodate a Wide Variety of Sulfation Patterns*
Jon A. Deakin
Cancer Research UK Glyco-Oncology Group, School of Cancer and Imaging Sciences, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Rd., Manchester M20 4BX, United Kingdom
Bärbel S. Blaum
Edinburgh Biomolecular NMR Unit, School of Chemistry, University of Edinburgh, West Mains Rd., Edinburgh EH9 3JJ, United Kingdom
John T. Gallagher
Dušan Uhrín
Malcolm Lyon
To whom correspondence should be addressed. Tel.: 161-446-3202; Fax: 161-446-3269
2 The abbreviations used are: HGF/SF, hepatocyte growth factor/scatter factor; AMAC, 2-aminoacridone; CHO, Chinese hamster ovary; dp, degree of polymerization, i.e. a dp4 corresponds to a tetrasaccharide; DS, dermatan sulfate; ERK, extracellular signal-regulated kinase; GAG, glycosaminoglycan; GalNAc, β-d-N-acetylgalactosamine; GlcNAc, α-d-N-acetylglucosamine; GlcN, α-d-N-substituted glucosamine; GMSA, gel mobility shift assay; HPLC, high performance liquid chromatography; HS, heparan sulfate; IdoA, α-l-iduronic acid; MAPK, mitogen-activated protein kinase; NK1, a truncated, splice variant of HGF/SF comprised of the N and first Kringle domains; NS, N-sulfate; SAX, strong anion-exchange; ΔHexA, Δ4,5-unsaturated hexuronate residue generated by lyase cleavage of hexosaminidic linkage; 2S, 2-O-sulfate; 4S, 4-O-sulfate; 6S, 6-O-sulfate.
3 D. Uhrin, unpublished data.
* This work was supported by a Cancer Research UK Programme Grant (to J. A. D., J. T. G., and M. L.) and an EaStCHEM Studentship (to B. S. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables I and II and Fig. 1.
Open AccessPublished:December 29, 2008DOI:https://doi.org/10.1074/jbc.M807671200
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Heparan sulfate (HS)/heparin and dermatan sulfate (DS) both bind with high affinity to hepatocyte growth factor/scatter factor (HGF/SF) and function as necessary co-factors in vitro. How both these two structurally distinct glycosaminoglycans (GAGs) are recognized has remained unclear. We have now reconciled this issue using a panel of minimal tri- and tetrasaccharide sequences of variable but well defined sulfation patterns in combination with further development of the gel mobility shift assay to allow simultaneous comparisons of relative protein affinities/selectivities for different oligosaccharides. From this approach it would seem that a minimum binding sequence is a disulfated trisaccharide comprised of an internal iduronate flanked by monosulfated hexosamine residues and that additional sulfation further enhances affinity. However, the similarity in recognition of HS/heparin and DS seems to arise primarily from a lack of any apparent positional requirement for sulfation. Thus, isomers of HS/heparin tetrasaccharides containing only two sulfates irrespective of whether they are purely N-, 2-O-, or 6-O-sulfates bind with equivalent apparent affinity as a disulfated DS tetrasaccharide. In addition, the NMR chemical shifts induced in NK1 (the truncated variant of HGF/SF comprised of the N-terminal and first Kringle domains) by titration with either heparin or DS oligosaccharides strongly indicate that both bind to essentially the same site. Together, these observations reveal an unexpected degree of flexibility in the GAG-HGF/SF interface, allowing a single binding site in the protein to accommodate iduronate-containing sequences of variable sulfation pattern and/or density from different GAGs.
Hepatocyte growth factor/scatter factor (HGF/SF)
The abbreviations used are: HGF/SF, hepatocyte growth factor/scatter factor; AMAC, 2-aminoacridone; CHO, Chinese hamster ovary; dp, degree of polymerization, i.e. a dp4 corresponds to a tetrasaccharide; DS, dermatan sulfate; ERK, extracellular signal-regulated kinase; GAG, glycosaminoglycan; GalNAc, β-d-N-acetylgalactosamine; GlcNAc, α-d-N-acetylglucosamine; GlcN, α-d-N-substituted glucosamine; GMSA, gel mobility shift assay; HPLC, high performance liquid chromatography; HS, heparan sulfate; IdoA, α-l-iduronic acid; MAPK, mitogen-activated protein kinase; NK1, a truncated, splice variant of HGF/SF comprised of the N and first Kringle domains; NS, N-sulfate; SAX, strong anion-exchange; ΔHexA, Δ4,5-unsaturated hexuronate residue generated by lyase cleavage of hexosaminidic linkage; 2S, 2-O-sulfate; 4S, 4-O-sulfate; 6S, 6-O-sulfate.
is a mesenchymal factor that provides growth, motility, and morphogenic stimuli to epithelial, endothelial, and neural cells via exclusive activation of the tyrosine kinase receptor MET (for review, see Ref.
Birchmeier C.
Birchmeier W.
Gherardi E.
Vande Woude G.F.
Nat. Rev. Mol. Cell. Biol. 2003; 4: 915-925
). There is increasing clinical interest in the potential therapeutic use of HGF/SF. Its natural role in wound healing and organ regeneration after injury provides opportunities for enhancement of its efficacy in tissue and organ repair (
Bevan D.
Fan T.P.
Edwards D.
Warn R.
J. Pathol. 2004; 203: 831-838
Chmielowiec J.
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Wehland J.
J. Cell Biol. 2007; 177: 151-162
). Conversely, its elevated or dysregulated activity in many solid tumors and lymphomas strongly correlates with invasiveness, metastasis, and poor disease prognosis and has highlighted its potential as a prominent target for therapeutic inhibition (
Corso S.
Comoglio P.M.
Giordano S.
Trends Mol. Med. 2005; 11: 284-292
Maulik G.
Shrikhande A.
Kijima T.
Ma P.C.
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Salgia R.
Cytokine Growth Factor Rev. 2002; 13: 41-59
Although the affinity of HGF/SF for MET is very high (KD of ∼0.2 nm) (
Mark M.R.
Lokker N.A.
Zioncheck T.F.
Luis E.A.
Godowski P.J.
J. Biol. Chem. 1992; 267: 26166-26171
), subsequent activation of the receptor leading to sustained and effective downstream signaling is highly dependent upon glycosaminoglycan (GAG) co-factors (
Deakin J.A.
Lyon M.
J. Cell Sci. 1999; 112: 1999-2009
Gallagher J.T.
J. Biol. Chem. 2002; 277: 1040-1046
Rubin J.S.
Day R.M.
Breckenridge D.
Atabey N.
Taylor W.G.
Stahl S.J.
Wingfield P.T.
Kaufman J.D.
Schwall R.
Botaro D.P.
Sergeant N.
Rudland P.S.
Fernig D.G.
Delehedde M.
Van der Voort R.
Taher T.E.I.
Wielenga V.J.M.
Spaargaren M.
Prevo R.
Smit L.
David G.
Hartmann G.
Pals S.T.
). Heparan sulfate (HS), heparin, and dermatan sulfate (DS), but not chondroitin sulfate, interact with HGF/SF with KD values in the range of 1–20 nm and function as co-factors for HGF/SF in vitro (
Deakin J.
Mizuno K.
Nakamura T.
Rahmoune H.
Biochemistry. 1998; 37: 6003-6008
J. Biol. Chem. 1998; 273: 271-278
Béchard D.
Gentina T.
Scherpereel A.
Aumercier M.
Vazeux R.
Richet C.
Degand P.
Jude B.
Janin A.
Tonnel A.-B.
Lassalle P.
). The in vitro activity of covalently cross-linked HGF/SF-GAG or -oligosaccharide conjugates (
) together with the affinity of heparin/HS for MET (
Lietha D.
Chirgadze D.Y.
Mulloy B.
Blundell T.L.
EMBO J. 2001; 20: 5543-5555
Youles M.E.
Miguel R.C.
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Gough J.
Bandyopadhyay A.
Butler P.J.G.
Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12039-12044
) suggests that a HGF/SF·GAG·MET ternary complex may be the effective signaling unit.
HGF/SF interacts with relatively short GAG oligosaccharides; the minimum even-numbered HS/heparin fragment that displays high affinity binding in vitro is a tetrasaccharide (dp4), and this also corresponds to the smallest, bioactive, repeating disaccharide structure (
Vidyasagar R.
McDonnell T.J.
). The major HS/heparin-binding site resides in the N-terminal domain of HGF/SF. Indeed, a heparin tetradecasaccharide has been co-crystallized with the truncated NK1 splice variant of HGF/SF, which only comprises the N-terminal domain, N, and the first Kringle domain, K1; the crystal structure identified the major GAG contacts as being in the N-domain, although in some crystals additional contacts were made with K1 (
). Subsequently, all the individually expressed domains of HGF/SF (i.e. the N-domain and the four Kringles, K1–K4, that comprise the α-chain and the serine proteinase homology domain, SP, corresponding to the β-chain) were compared by heparin affinity chromatography and gel mobility shifts assays, and these confirmed the existence of a weaker GAG-binding site in K1 as well as revealing an additional low affinity site in the SP domain (
Holmes O.
Pillozzi S.
Carafoli F.
Kemp L.
J. Mol. Biol. 2007; 367: 395-408
). However, there are also other reports using domain-deleted (
Prospero T.
Brinkmann V.
Ozcelik O.
Winter G.
Hepple J.
Batley S.
Bladt F.
Sachs M.
Curr. Biol. 1997; 8: 125-134
) and point-mutated (
Inoue H.
Hagiya M.
Shimizu S.
Nose T.
Shimohigashi Y.
) forms of HGF/SF that suggest that K2 may contribute to GAG binding by native, full-length HGF/SF even though this was not confirmed by the behavior of isolated K2 (
NK1 also binds DS (
), probably mediated by the N-domain (
); neither the isolated K1 domain nor the β-chain from HGF/SF bind independently to DS even though they possess weak heparin affinity (
). Thus, joint heparin/HS and DS affinity appears to be restricted to the N-domain. This begs the question as to whether both GAG types bind with high affinity to a single, dual-specificity binding site or whether the N-domain contains two distinct sites with differential GAG specificities.
A recent investigation of the binding and activating properties of a range of native and specifically desulfated GAGs suggested that GAG binding to NK1 was of relatively low specificity (
Catlow K.R.
Wei Z.
Pavaño M.S.G.
). Binding could be sustained by a low level of overall sulfation (as displayed by DS) as long as, critically, the GAG contained iduronate (IdoA), although increased sulfation enhanced affinity (as with heparin) and ultimately could override the need for IdoA. The greater conformational flexibility of IdoA is assumed to provide increased overall chain flexibility that may optimize the spatial disposition of the necessary contact groups within the GAG. A low sulfate density in the presence purely of β-D-glucuronic acid, as in mammalian chondroitin sulfate, was inadequate for binding NK1. The results of selective desulfations suggested that there may not be a strict requirement for any specific sulfation position in either HS/heparin or DS. This is compatible with the possibility of the N-domain containing a single low specificity binding site for both HS/heparin and DS. However, interpretations of such studies using polymeric GAGs are complicated because desulfation methods are rarely quantitative, and minor amounts of specific sulfate groups may be sufficient to support binding especially if the protein recognizes short sequences.
The GAG co-factor requirement of HGF/SF-mediated activation of MET provides an opportunity for potential clinical inhibition of HGF/SF activity via disruption of the necessary component co-factor interaction (for review, see Ref.
Delehedde M. Lortat-Jacob H. New Developments in Therapeutic Glycomics. Research Signpost, Trivandrum, India2007: 317-350
). The ability of exogenous GAG species as small as dp4s to engage in productive complex formation opens up possibilities for small-molecule inhibition either based upon potentially inhibitory, modified GAG oligosaccharides or novel synthetic mimetics. Our recent demonstration of the inhibition of HGF/SF-mediated ERK-MAPK signaling and cell migration in vitro by synthetic, sulfated, non-sugar GAG mimics (
Raiber E.-A.
Wilkinson J.A.
Manetti F.
Botta M.
Ducki S.W.
Bioorg. Med. Chem. Lett. 2007; 17: 6321-6325
) demonstrated proof of principle and opens the way for improved inhibition by better-designed agents.
It is difficult to elucidate the GAG binding specificity of HGF/SF using intact, structurally heterogeneous GAGs. Thus, the aims of the present study were to utilize minimal GAG oligosaccharides of defined structure (see supplemental Tables 1 and II) to (i) provide an improved description of the structural requirements for GAG-HGF/SF interaction and also (ii) to resolve the question of whether HS/heparin and DS occupy identical or distinct binding sites within the N-domain of HGF/SF. Such information may aid the future rational design of small-molecule inhibitors of HGF/SF that target its GAG co-factor requirement.
Materials-Recombinant human HGF/SF was obtained from R&D Systems (Abingdon, UK). Bovine lung heparin, porcine intestinal mucosal heparin, chondroitinases ABC (Proteus vulgaris; EC 4.2.2.4), and chondroitinase AC-I (Pedobacter heparinus; EC 4.2.2.5) were obtained from Sigma. Low molecular weight heparin (Innohep®) was from Leo Pharma (Princes Risborough, UK). Porcine intestinal mucosal DS was from Celsus Laboratories Inc. (Cincinatti, OH). Completely de-sulfated/re-N-sulfated heparin was obtained from Seikagaku Corp. (Tokyo, Japan). Heparinases I (P. heparinus; EC 4.2.2.7), II (P. heparinus; no EC number assigned), and III (P. heparinus; EC 4.2.2.8) were purchased from Grampian Enzymes (Harray, Orkney, UK). Bio-Gel P10 was from Bio-Rad Laboratories. Pre-packed PD-10 desalting columns were purchased from GE Healthcare. The CHO pgsA-745 mutant cell line was a generous gift from Prof. Jeffrey Esko (University of California at San Diego, CA).
Preparation of NK1-Human NK1 was purified from the culture medium of a Pichia pastoris recombinant expression system provided by Dr. Ermanno Gherardi (MRC Centre, Cambridge, UK) as previously described (
Hepple J.P.
Zhou H.
Byrd A.
Nat. Struct. Biol. 1999; 6: 72-79
). 15N-Labeled NK1 for NMR titrations was produced by growth of the P. pastoris clone in 0.8-liter cultures in a Bioflow 3000 fermentor (New Brunswick Scientific, St. Albans, UK) using [15N]ammonium sulfate (>98%; Sigma) as the sole nitrogen source.
Preparation of Hexasulfated Heparin Tetrasaccharide- Heparin dp4s were prepared by the fractionation of low molecular weight heparin by gel filtration. To increase the yield of dp4s, the heparin was partially digested with heparinase I (0.1 unit added to a 60-ml solution of 5 mg/ml heparin in 0.1 m sodium acetate, 0.1 mm calcium acetate, pH 7.0, at 37 °C for 6 h). Small aliquots were removed at regular intervals and rapidly chromatographed on a Superdex Peptide HR 10/30 HPLC column (10 × 300 mm; GE Healthcare Life) eluted with 0.2 m NH4HCO3 at 0.5 ml/min and monitored by UV absorbance at 232 nm to assess the degree of depolymerization. At an appropriate level of breakdown, the digest was terminated by boiling, concentrated by lyophilization, and then subjected to gel filtration chromatography on a Bio-Gel P-10 column (2.5 × 115 cm) eluted with 0.2 m NH4HCO3 at a flow rate of 10 ml/h. Fractions of 2 ml were collected and monitored for UV absorbance at 232 nm. The peak corresponding to total dp4s was pooled and freeze-dried twice to remove NH4HCO3. This was then subfractionated by application in pH 3.5 water (adjusted with HCl) to a semipreparative ProPac PA1 strong anion-exchange (SAX) HPLC column (4.6 × 250 mm; Dionex, Camberley, UK) eluted with a gradient of 0–1.5 m NaCl, pH 3.5, over 90 min at a flow rate of 4 ml/min with in-line monitoring of UV absorbance at 232 nm. The latest eluting dp4 peak, corresponding to the hexasulfated species, was collected and desalted on a PD10 column run in water. Quantitation was by absorbance at 232 nm. Composition was confirmed by disaccharide analysis.
Preparation of DS Tetrasaccharides-Porcine intestinal mucosal DS (1 g) in 50 ml of 75 mm NaCl, 10 mm sodium phosphate, pH 7.0, was fully digested with 1 IU of chondroitinase AC-1 followed by partial digestion with 1 IU of chondroitinase ABC. Small aliquots were removed at regular intervals and checked for the degree of breakdown as described earlier. The final digest was fractionated on a Bio-Gel P-10 column (as described for heparin oligosaccharides above), and the dp4 peak was pooled and freeze-dried twice to remove NH4HCO3.
The mixed DS dp4 pool was applied in water to a Sphereclone 5-μm SAX-HPLC column (4.6 × 250 mm; Phenomenex, Macclesfield, UK) and eluted with a linear gradient of 0–1.0 m NaCl over 60 min at a flow rate of 1 ml/min. UV absorbance at 232 nm was monitored on-line. Individual dp4 peaks were separately pooled, desalted on PD-10 columns eluted with distilled water, and then freeze-dried. Quantitation was by absorbance at 232 nm. Compositions and, thus, likely structures were confirmed by disaccharide analyses.
Preparation of Heparin and DS Trisaccharides-Trisaccharides (dp3s) were prepared by chemical removal of the non-reducing unsaturated uronate residue from heparin, and DS dp4s by the method of Ludwigs et al. (
Ludwigs U.
Elgavish A.
Esko J.D.
Meezan E.
Rodén L.
Biochem. J. 1987; 245: 795-804
). dp4S (1 mg) were dissolved in 75 μl of 10 mm mercuric acetate, pH 5.0, and incubated at 20 °C for 40 min. The sample was adjusted to 1 ml with 5 m NaCl and applied to a Superdex Peptide HR 10/30 HPLC column equilibrated in 0.2 m NH4HCO3. The column was run at a flow rate of 0.5 ml/min with on-line UV monitoring at 206 nm. Fractions of 0.25 ml were collected, and the peak corresponding to the dp3 was collected and freeze-dried twice to remove NH4HCO3.
Preparation of Heparin Tetrasaccharides Desulfated at a Single Specific Ring Position-Hexasulfated heparin dp4 was subjected to specific desulfations at either 2-O, 6-O, or N positions using the procedures previously described for intact heparin (
). The specifically desulfated dp4s were separated from partially and over-desulfated species on a semipreparative ProPac PA1 SAX-HPLC column as described earlier. Individual species were desalted on PD10 columns, and their compositions were checked by disaccharide analysis.
Preparation of N-Sulfated Heparin Tetrasaccharide-Completely de-N/O-sulfated/re-N-sulfated heparin in 50 mm sodium acetate, 0.5 mm calcium acetate, pH 7.0, was digested with 0.2 IU/ml each of heparinases I, II, and III. Digestion was allowed to proceed at room temperature until the resulting dp2 and dp4 species constituted more than 50% of the total products (as assessed by chromatography of an aliquot upon a Superdex Peptide column, as described earlier). The dp4s, recovered by Superdex Peptide gel filtration, were freeze-dried and subjected to disaccharide compositional analysis.
Preparation of 2-O- or 6-O-Sulfated Heparin Tetrasaccharides-Porcine intestinal heparin was subjected to either (i) de-6-O sulfation, with concomitant de-N sulfation, followed by re-N acetylation or (ii) de-2-O sulfation followed by sequential de-N sulfation and re-N acetylation, essentially as described in Catlow et al. (
). Each product was partially digested with heparinases, and the dp4 pool was collected by gel filtration and then subfractionated by SAX-HPLC as described for singly desulfated heparin dp4s (above). Compositions were checked by disaccharide analyses.
Disaccharide Analysis of GAGs and Oligosaccharides-DS and heparin species were completely digested to disaccharides using chondroitinase ABC or a mixture of heparinases I, II, and III. The resulting disaccharides were separated on ThermoQuest Hypersil SAX-HPLC (4.6 × 240 mm; ThermoQuest Hypersil Division, Runcorn, UK) or ProPac PA1 SAX-HPLC (4 × 250 mm; Dionex) columns as previously described (
). Columns were calibrated with known disaccharide standards.
Fluorescent Labeling of Oligosaccharides and Gel Mobility Shift Assays-Oligosaccharides were tagged at their reducing ends with 2-aminoacridone (AMAC), as described in Lyon et al. (
). For gel mobility shift assays (GMSA), AMAC-tagged oligosaccharides were preincubated with NK1 protein in a 1:1 ratio for 30 min in a total volume of 10 μl of phosphate-buffered saline. Samples were electrophoresed on either agarose gels (1% (w/v)) or polyacrylamide gels (6% (w/v) for basic GMSA or 10% (w/v) for selection GMSA) at a constant 100 V for between 5 and 20 min (shorter times for basic GMSA but often longer for selection GMSA) and then imaged under UV light (
). In selection GMSA experiments, NK1 is titrated against a fixed concentration of a mixture of two or more oligosaccharides (of equivalent molar concentrations); the molar concentration of NK1 is expressed relative to the total ligand concentration (i.e. a NK1/ligand ratio of 0.33 for a mixture of three ligands indicates that the NK1 molar concentration is equal to that of any single ligand species in the mixture).
Reverse-phase Chromatography of AMAC-labeled Oligosaccharides-Bands of free AMAC oligosaccharides were excised from 1% agarose GMSA gels with a scalpel blade under UV visualization. Gel fragments were mixed end-over-end for 2 h in 1 ml of distilled water in a sealed tube to extract the bulk of the oligosaccharides. The supernatant was then freeze-dried, redissolved in 20 μl of water, and then applied to an Eclipse XDB C-18 reverse-phase HPLC column (4.6 × 150 mm; Agilent Technologies, Stockport, UK) pre-equilibrated in 0.1 m ammonium acetate (Solution A). After a short and steep 2-ml gradient of 0–10% solution B (100% methanol), the oligosaccharide components were resolved over a 50-ml linear gradient of 10–30% solution B at a flow rate of 1 ml/min, with in-line monitoring of AMAC fluorescence (excitation at 425 nm, emission at 520 nm).
Assay for Activation of ERK-MAPK in GAG-deficient Cells- GAG-deficient CHO pgsA-745 mutant cells were cultured as previously described (
). Cells were serum-starved for 2 h before treatment with HGF/SF with or without oligosaccharides (when combined, they were preincubated for 30 min before the addition to the cells). Downstream signaling was assayed 20 min after reagent addition. Cell lysate preparation, SDS-PAGE, and Western blotting using a mouse monoclonal antibody against dual-phosphorylated (Thr183/202/Tyr185/204) ERK-1/2 MAPKs (Santa Cruz Biotechnology Inc., Santa Cruz, CA) were performed as previously described (
NMR Titrations-Concentrations of 15N-labeled NK1 used in the GAG titrations were 50 and 44 μm when titrated with heparin dp4 and DS dp6, respectively. Titration points were recorded at protein-oligosaccharide ratios of 1:1 and 1:2 for heparin dp4, whereas for the DS dp6 the ratios used were 1:0.5, 1:1, and 1:2.5. All spectra were recorded in 20 mm sodium acetate, 100 mm sodium chloride, pH 6.0, at 30 °C on an 800-MHz Avance NMR spectrometer. Spectra were processed using AZARA (Version 2.7, Wayne Boucher and Dept. of Biochemistry, University of Cambridge), and the chemical shift changes were quantified using ANALYSIS (
Vranken W.F.
Boucher W.
Stevens T.J.
Fogh R.H.
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Ulrich E.
Markley J.L.
Ionides J.
Laue E.D.
Proteins. 2005; 59: 687-696
NK1 Binding to Heparin and DS Tetrasaccharides-Previous studies had reported that a dp4 was the smallest even-numbered heparin oligosaccharide with affinity for HGF/SF and NK1 (
). By contrast, a dp6 appeared to be the minimal DS oligosaccharide (
). This size difference in the recognition of these two GAGs has now been revisited.
dp4s were generated from heparin by limited heparinase I digestion followed by gel filtration chromatography (Fig. 1A). The major dp4 species, corresponding to the hexasulfated ΔHexA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS(6S), was further purified by SAX-HPLC (Fig. 1C), and its composition and sequence were confirmed by disaccharide analysis and NMR (not shown). A porcine intestinal mucosal DS preparation with a high IdoA content (indicated by its substantial resistance to chondroitinase ACI digestion) but only a low level of 2-O sulfation of these IdoA residues was partially fragmented by complete chondroitinase ACI digestion followed by partial chondroitinase ABC scission. The dp4 pool was recovered by gel filtration chromatography (Fig. 1B). Fractionation of this pool by SAX-HPLC (Fig. 1D) yielded a major disulfated species (∼75% total) that disaccharide analysis revealed to be comprised predominantly of 4-O-sulfated disaccharides (92.5% 4-O-sulfates and 7.5% 6-O-sulfates, with no detectable 2-O-sulfates), i.e. corresponding primarily to the sequence ΔHexA-GalNAc(4S)-IdoA-GalNAc(4S).
FIGURE 1Preparation of GAG tetrasaccharides. dp4s were prepared from both heparin (A and C) and DS (B and D) by limited enzymic digestions, gel filtration on Bio-Gel P-10 (A and B), and then SAX-HPLC (C and D) as described under "Experimental Procedures." In C, the latest-eluting peak (a) is the hexasulfated heparin dp4, whereas the earlier-eluting peak (b) is the corresponding pentasulfated dp4 lacking a 6-O-sulfate at the reducing terminal. In D the major peak (a) corresponds to the di-4-O-sulfated DS dp4, and the later-eluting minor peak (b) corresponds to a trisulfated dp4 that also bears a single 6-O-sulfate.
Both heparin and DS dp4s were labeled with AMAC, and their interaction with NK1 was assessed by GMSA. As previously demonstrated (
), the heparin dp4 bound strongly to NK1 and was retained in the well (Fig. 2A). The DS dp4 also bound but required higher concentrations of NK1 than the heparin (Fig. 2B) and higher than used in the previous study (
). Thus, an equivalent minimal size of heparin/HS and DS support binding to NK1, although affinity is appreciably higher for a heparin-derived species.
FIGURE 2Binding of NK1 to GAG tetrasaccharides. The interactions of NK1 with 1 μg of hexasulfated heparin dp4 (A) or disulfated DS dp4 (B) were assessed by direct GMSA on either a 6% polyacrylamide gel (A) or a 1% agarose gel (B) as described under "Experimental Procedures." NB, the direction of migration is shown as downward in A but upward in B. Binding of the fluorescent AMAC-oligosaccharide is shown by depletion of the mobile free species, and the reciprocal appearance of a relatively immobile complex with NK1 in or close to the well (note that sometimes this can be lost from the well during gel handling).
The original unfractionated DS dp4 pool also contains a small proportion (∼7%) of a trisulfated species (Fig. 1D) that, from disaccharide analysis, is comprised of equal proportions of HexA-GalNAc(4S) and a disulfated disaccharide that is primarily HexA-GalNAc(4S,6S) (much lesser amounts of HexA(2S)-GalNAc(4S) and HexA(2S)-GalNAc(6S) were also present). Based on its complete resistance to chondroitinase ACI (not shown), the predominant sequence of this trisulfated dp4 is likely to be ΔHexA-GalNAc(4S,6S)-IdoA-GalNAc(4S).
To directly compare the relative affinities of NK1 for dp4s that differ in sulfate densities and/or polymer backbones, it is possible to perform a GMSA experiment in a new mode, which we refer to as a "selection" mode. In this new variant the protein is incubated simultaneously with a mixture of equimolar amounts of the different AMAC-tagged dp4s. If the latter differ in sulfate density they will, as free species, resolve into separate bands on the GMSA gel. Thus, if for example the species differ very markedly in protein affinity, then there is sufficient protein present to selectively and almost quantitatively bind and deplete the highest affinity species while leaving the other, low affinity species relatively unaffected (though the latter will progressively bind if the ratio of protein is increased). Alternatively, if all species possess very similar protein affinities, then all bands should become partially depleted in parallel as the protein content is increased. The overall pattern of selective or non-selective band depletion can, therefore, be interpreted as a reflection of relative NK1 affinities.
Comparison of the disulfated and trisulfated DS dp4s by selection GMSA revealed a clear preferential affinity for the latter, more highly sulfated species (Fig. 3A). When the experiment is extended to also include the hexasulfated heparin dp4, then it is clear that the latter is greatly preferred over both DS species (Fig. 3B). Thus, the order of relative dp4 affinities is hexasulfated heparin >> trisulfated DS > disulfated DS.
FIGURE 3Relative affinities for NK1 of different GAG tetrasaccharides. The interactions of NK1 with mixtures of AMAC-labeled disulfated (a) and trisulfated (b) DS dp4s (A) or the latter together with the hexasulfated heparin dp4 (c) (B) were assessed by selection GMSA on a 10% polyacrylamide gel as described under "Experimental Procedures." An increasing molar ratio of NK1 was titrated against a fixed amount of mixed dp4s containing 1 μg of each species. Note the increased, selective depletion of species b and c in panels A and B, respectively.
NK1 Binding to Heparin and DS Trisaccharides-Previously, only even-numbered oligosaccharides, generated by enzymic scission of polymers with repeating disaccharide structures, have been tested for NK1 affinity. Odd-numbered oligosaccharides can be generated by mercuric ion-catalyzed removal of the unsaturated hexuronate from the non-reducing end of such even-numbered species. There is no corresponding procedure for alternative monosaccharide removal at the reducing end. Truncation at the non-reducing end will at most result in the loss of only one sulfate group (i.e. a2-O-sulfate) and often none at all. Thus, we decided to prepare and test the affinity of the respective pentasulfated and disulfated dp3s, i.e. GlcNS(6S)-IdoA(2S)-GlcNS(6S) and GalNAc(4S)-IdoA-GalNAc(4S), which could be generated from the major hexasulfated heparin and disulfated DS dp4s described earlier. In addition, the tetrasulfated heparin dp3, GlcNS(6S)-IdoA(2S)-GlcNS, was prepared from the pentasulfated dp4, ΔHexA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS, a less common species in the heparin dp4 pool (Fig. 1B) whose composition and sequence was previously determined by disaccharide analysis and NMR (
Jin L.
Barran P.E.
Uhrín D.
Phys. Chem. Chem. Phys. 2005; 7: 3464-3471
). In all cases removal of the unsaturated hexuronate residue from the non-reducing end could be confirmed by any of a variety of techniques, i.e. smaller size and, thus, later elution on a Superdex Peptide column (with concomitant loss of absorption at 232 nm, so requiring monitoring of the dp3 at 206 nm), an altered size and charge density that affects electrophoretic mobility on a polyacrylamide gel, or changed elution properties on a C18 reverse-phase HPLC column (not shown).
Direct GMSA assays to test individually the two heparin dp3s showed that they also both bind to NK1 (Fig. 4A). Binding of the DS dp3 (that has lost a hexuronate from the non-reducing end, but no sulfation) required a higher ratio of NK1-oligosaccharide, as with the parent dp4, and their affinities appear to be broadly similar (not shown).
FIGURE 4Binding of NK1 to GAG trisaccharides. A, binding to NK1 by direct GMSA on a 6% polyacrylamide gel of the tetrasulfated (3b) and pentasulfated (3a) heparin dp3s compared with their parent pentasulfated (4b) and hexasulfated (4a) dp4s. B, selection GMSA using an increasing molar ratio of NK1 titrated against a fixed amount of a mixture (1 μg of each species) of hexasulfated heparin dp4 (4a) and its derived pentasulfated dp3 (3a) resolved on a 10% polyacrylamide gel. C, selection GMSA using an increasing molar ratio of NK1 titrated against a fixed amount of a mixture (1 μg of each species) of pentasulfated heparin dp3 (3a), disulfated DS dp4 (4a), and trisulfated DS dp4 (4b) separated on a 10% polyacrylamide gel.
In the latter case both dp3 and dp4 species are disulfated; consequently, their migration on PAGE is almost identical, and thus, direct comparison of affinities by selection GMSA could not be performed at that time. However, selection GMSA to compare the hexasulfated heparin dp4 versus its slightly faster-moving pentasulfated dp3 derivative (i.e. lost a 2-O-sulfated hexuronate from the non-reducing end) shows a clear preference by NK1 for the dp4 species (Fig. 4B). In contrast, selection GMSA in which the pentasulfated dp3 from heparin is compared with both the di- and tri-sulfated dp4s from DS reveals that NK1 still retains a preference for the heparin ligands even though the DS ones are longer (Fig. 4C). Affinity is, thus, not primarily dependent upon ligand length but also involves aspects of sulfate density and/or GAG backbone. A previous study had revealed the importance of IdoA for GAG binding (
), and the binding of dp3s indicates that only a single internal IdoA is actually required.
Effects of Single and Double, Site-specific Desulfations of Heparin Tetrasaccharides upon NK1 Binding-Heparin oligosaccharides have a higher NK1 affinity than same-sized DS oligosaccharides, which may reflect the higher sulfate densities present in the former. Heparin oligosaccharides can be potentially N-, 2-O-, and 6-O-sulfated. To test whether the position of sulfation has any significant influence upon NK1 binding, we specifically desulfated the hexasulfated heparin dp4 to yield the three alternative tetrasulfated dp4s (i.e. 2-O-/6-O-, N-/2-O-, and N-/6-O-sulfated) that completely lack sulfation at one of the three specific positions. The advantage of working with dp4s is that subsequent SAX-HPLC separation (with confirmation of composition by disaccharide analysis) allows the desired, homogeneous, fully modified species to be selectively purified from any non- or partially desulfated species or from species that may have experienced additional, nonspecific changes. This contrasts with the experience of working with desulfated GAG chains where complete specific desulfation is rarely achieved, and nonspecific effects can also accumulate to varying degrees, leading to chain heterogeneities that potentially complicate affinity interpretations (
GMSA analysis of NK1 binding to each of these three tetrasulfated heparin dp4s showed that they all bind with high affinity to NK1 (Fig. 5). Thus, importantly, heparin/HS binding to NK1 is not critically dependent upon the presence of sulfation at any one specific position within the repeating disaccharide unit, at least if both other major positions are fully sulfated.
FIGURE 5Binding of NK1 to heparin tetrasaccharides lacking sulfation at a specific position. Binding of NK1 (20 μg) by direct GMSA on a 6% polyacrylamide gel to tetrasulfated heparin dp4s (0.25 μg) that specifically lack N-sulfates, 2-O-sulfates, or 6-O-sulfates.
Clearly, only two sulfate groups are sufficient to elicit relatively weak binding of a DS dp3 or dp4 to NK1, although heparin species that naturally possess higher levels of sulfation (and variable positioning) bind NK1 with greater affinity. Would a heparin-derived dp4 that possesses an equivalent sulfate density to a DS dp4 also possess a similar NK1 affinity?
To address this issue we specifically desulfated heparin at all three combinations of two specific positions, i.e. leaving sulfation at just one remaining, but specific position (i.e. an N-, 2-O-, or 6-O-sulfate) in each disaccharide repeat, as are often found in HS sequences. These were then partially fragmented using heparinases, and the dp4 pools were recovered from the digests by gel filtration and then fractionated by SAX-HPLC. The three distinct, homogeneous disulfated dp4s were, thus, recovered as pure species, their compositions were confirmed by disaccharide analyses, and their NK1 affinities were tested by GMSA.
All three disulfated heparin dp4s bound NK1 by direct GMSA, broadly similar to the behavior of a disulfated DS dp4 (Fig. 6A and Table 1). Note that the affinity for the DS dp4 appears to be greater here than was originally revealed in Fig. 2. This is probably because a relatively old batch of NK1 was used for the latter, whereas all subsequent experiments used a fresh batch whose concentration of effective, monomeric protein was likely to be higher.
FIGURE 6Binding of NK1 to heparin tetrasaccharides sulfated solely at a specific position. A, binding of NK1 (20 μg) by direct GMSA on a 1% agarose gel to three disulfated heparin dp4s that contain sulfates at only one position within the disaccharide repeat (i.e. either N-sulfates, 2-O-sulfates, or 6-O-sulfates) and also the di-4-O-sulfated DS dp4. On the right-hand side of the gel the GMSA is performed with an equimolar mix of the four dp4s above (1 μg each) titrated against an increasing molar ratio of NK1. B, the bands in A corresponding to the mixtures of non-complexed AMAC-labeled dp4s (labeled in the figures as free dp4s) from the titration with NK1 were excised, and the dp4 species were eluted and then resolved by reverse-phase HPLC (the elution gradient is only shown on the bottom panel for clarity) as described under "Experimental Procedures." Fluorescence peak areas were quantified, and the relative proportions of each dp4 species present are presented in Table 1. Note that equimolar amounts of the various dp4 species were used though the efficiency of AMAC labeling, and thus, the fluorescence yield, varied between individual species as can be seen from the relative band intensities in A and peak areas in the top panel of B.
TABLE 1Relative abundances of the four different dp4 species present in the free dp4 pool The band corresponding to the free dp4 pool was extracted from a selection GMSA gel (Fig. 6A), and the four constituent disulfated dp4s were resolved by reverse phase HPLC (Fig. 6B). The peak areas were calculated for each NK1:GAG ratio and then expressed as % of that originally present in the absence of NK1.
NK1/total dp4 ratio
Di-N-sulfated heparin dp4
Di-6-O-sulfated heparin dp4
Di-4-O-sulfated DS dp4
No NK1 100 100 100 100
1:1 70.2 82.4 68.0 70.9
4:1 4.7 4.6 4.6 5.6
To detect any preferential NK1 binding affinity within these dp4s it would be useful to perform GMSA in the selection mode as used earlier. Unfortunately, all three heparin dp4s as well as the DS dp4 are disulfated and, thus, have the same charge/mass ratio and consequently do not separate from each other on GMSA gels. When NK1 is incubated with a mixture of equimolar amounts of all four dp4s and run on a GMSA gel, a single combined band of free dp4s is seen which becomes increasingly depleted as the ratio of NK1 is increased, confirming that all four species can bind (Fig. 6A). However, in such a scenario it is possible to excise the fluorescent band of free dp4s from the gel, elute them by diffusion, and subsequently separate them into their four constituent dp4s by reverse-phase HPLC with fluorescent detection (as recently developed by us for disaccharide analysis of GAGs (
Glycobiology. 2008; 18: 483-491
)). It is clear from such an analysis that as NK1 is increased, leading to the recruitment of a greater proportion of the total dp4s into complexes, the increasingly depleted pool of free dp4s still retains an approximate equimolar ratio of the four constituent dp4s (Fig. 6B). A more simplified selection GMSA in which only the di-N-sulfated heparin dp4 was compared with the di-4-O-sulfated DS dp4 confirmed this finding (not shown). As the free dp4 pool was progressively reduced (from 100 to 53, 36, and finally, 18% of total pool) by an increasing amount of NK1, the ratio of heparin dp4 to DS dp4 remained almost constant throughout (ratios of 1.05, 1.11, and 1.05, respectively). Not only is there no discrimination between the three different HS/heparin dp4s but NK1 does not now select preferentially for the HS/heparin over the DS species. Thus, similar NK1 affinities for these disulfated dp4s are essentially dictated by their equivalent sulfate densities and are independent of both GAG backbone and sulfate positioning.
Stimulation of ERK-MAPK Phosphorylation by HGF/SF in Response to Tri- and Tetrasaccharides-Various dp3 and dp4 species were tested for their ability to elicit HGF/SF activity in the CHO pgsA-745 mutant cell line, as measured by dual phosphorylation of ERK-MAPK. These cells possess MET but are lacking in sulfated GAGs, being defective in the xylosyltransferase normally responsible for initiating serine-linked synthesis of the GAG chains (
Stewart T.E.
Taylor W.H.
Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201
). Consequently, these cells are barely responsive to HGF/SF unless GAG chains or oligosaccharides are provided exogenously. It has previously been shown that native heparin/HS dp4s can provide a co-factor stimulus, although a much weaker one than elicited by intact heparin: ∼10-fold less ERK-MAPK phosphorylation in CHO pgsA-745 cells (
) and ∼100-fold less DNA synthesis in sulfated GAG-deficient HaCaT cells (
). Comparisons of various dp4 species show that the di- and trisulfated DS dp4s as well as all three variants of the disulfated heparin dp4s elicit broadly similar levels of ERK-MAPK phosphorylation (Fig. 7). However, both the heparin and especially the DS dp3 species gave weaker responses than the dp4s (Fig. 7). This suggests that the additional GAG-NK1 contacts made by a longer ligand may help significantly in generating a signaling response, especially with lower sulfated species.
FIGURE 7Activation of HGF/SF-MET signaling by tri- and tetrasaccharides. Downstream activation (dual phosphorylation) of ERK-MAPK in GAG-deficient CHO pgsA-745 cells in response to HGF/SF (25 ng/ml) with or without various dp3 and dp4 species (1 μg/ml) derived from heparin and DS. The heparin oligosaccharides are hexasulfated dp4 (4a), pentasulfated dp3 (3a), di-N-sulfated dp4 (N), di-6-O-sulfated dp4 (
), di-2-O-sulfated dp4 (
). The DS oligosaccharides are trisulfated dp4 (4b), disulfated dp4 (4a), disulfated dp3 (3a). Quantitation of relative phosphorylation levels by densitometry (normalized to a value of 1.0 for the control of HGF/SF alone) gave values of 3.7, 3.0, 5.8, 6.2, 5.9, 8.8, 4.8, and 1.6 for the eight oligosaccharides in the order given above.
NMR Chemical Shift Titrations of NK1 with Heparin and DS Oligosaccharides-We additionally addressed the issue of GAG-binding site specificity at the level of the protein NMR spectrum and performed a series of titrations of 15N-labeled NK1 with hexasulfated heparin dp4 in comparison with the disulfated DS dp4 and the trisulfated DS dp6. The heparin dp4 displayed the typical behavior of a strong binder. The addition of an equimolar amount of dp4 ligand resulted in the disappearance of some of the 15NH cross-peaks of NK1, concomitant with their reappearance at new positions, whereas other signals were unaffected (supplemental Fig. 1). Increasing the molar ratio of GAG/NK1 from 1:1 to 2:1 did not cause further cross-peak movements, implying near-saturation of binding sites at equimolar concentrations. In contrast, titrating DS dp4 with NK1 up to a molar excess of 2.5:1 resulted in chemical shift changes that only occasionally exceeded the natural line widths of NK1 cross-peaks (data not shown), indicating much weaker binding. Nevertheless, the affected peaks were the same as those which experienced large movements upon titration with heparin dp4. This suggests that the binding sites for both dp4s overlap.
We have shown in this study and previously (
) that DS dp6 binds NK1 more strongly than the corresponding DS dp4. We, therefore, titrated increasing amounts of DS dp6 into NK1 up to a 2.5:1 molar ratio. Binding was fast on the NMR time scale, resulting in characteristic gradual movements of NH cross-peaks upon increasing additions of dp6 (supplemental Fig. 1). These movements were much more pronounced than observed with DS dp4, confirming the stronger binding of the dp6. Even more revealing was the fact that a substantial number of cross-peaks in the DS dp6 titration move toward the cross-peaks observed for the NK1-heparin dp4 complex (Fig. 8A; supplemental Fig. 1), indicating similar chemical environments of affected residues in complexes with both the heparin dp4 and DS dp6.
FIGURE 8NMR chemical shift titrations of NK1 with oligosaccharides. A, a selected portion of the more extensive 1H,15N heteronuclear single quantum correlation spectra (supplemental Fig. 1) showing the chemical shifts of 15NH cross-peaks in NK1 (black) induced by 1:1 titrations with either heparin dp4 (blue) or DS dp6 (orange). Three identified residues, Cys-70, Arg-73, and Leu-80 demonstrate shifts (connected by a line) elicited by both oligosaccharides but consistently greater with the heparin dp4 than with the DS dp6. B, comparative histogram of combined chemical shift changes. Shift changes observed upon the titrations with heparin dp4 (blue) or DS dp6(orange) were calculated using
Δδ=δ1H2+δ15N52,
where δ1H and δ15N are the chemical shift changes in the 1H and 15N dimensions, respectively. Changes are ranked in the order of declining magnitude of Δδ observed for different NH cross-peaks in the titration with heparin dp4 (supplemental Fig. 1). The shift changes observed with the DS dp6 are then superimposed in the same order of NH cross-peaks.
Although tracking the chemical shift changes of residues during titrations was hindered by overlap and peak broadening in some regions, it was nevertheless possible to perform a qualitative comparison of the heparin dp4 and DS dp6 interactions. Where possible, the combined 1H and 15N chemical shift changes in NK1 elicited by heparin dp4 were calculated and then sorted in decreasing order of magnitude. The corresponding changes observed upon titration with the highest ratio of DS dp6 were then overlaid in the same order of cross-peaks (Fig. 8B). This comparison reveals major similarities between the two titrations, consolidating the evidence that HS/heparin- and DS-binding sites in NK1 overlap. For the majority of the cross-peaks, the absolute changes were larger for the heparin dp4 than for the DS dp6 at the same protein/ligand ratio, and indeed the NMR confirms the relative order of binding affinity for NK1 as heparin dp4 > DS dp6 >> DS dp4. However, a fraction of the shifting cross-peaks observed in the DS dp6 titration exceeded the corresponding changes caused by the heparin dp4, as maybe would be expected for the different nature and/or sizes of the two oligosaccharide ligands.
The known ability of both heparin/HS and DS to interact strongly with HGF/SF and to act with similar potencies as co-factors in its activity is a relatively unusual property among "heparin-binding" proteins. Such proteins generally show greater GAG selectivity, i.e. relatively weak affinities for DS compared with heparin/HS.
Proteins with a less strict GAG specificity potentially pose greater problems in elucidating their recognition properties than might be faced when dealing with a highly specific protein like for example antithrombin-III, which recognizes an unusual heparin/HS dp5 sequence and tolerates relatively little variation (
Thunberg L.
Bäckström G.
Lindahl U.
Carbohydr. Res. 1982; 100: 393-410
). Working with structurally heterogeneous, intact GAGs or their incompletely desulfated variants often provides relatively limited information, especially if only short sequences are sufficient for protein recognition. Ultimately, definitive information can only emerge from the use of rigorously defined structures; hence, our present study of the minimal sizes of GAG oligosaccharides required for NK1 binding and the consequent effects of precise positional changes in their sulfate patterning.
GMSA analyses revealed that dp3s/dp4s are the minimal odd- and even-numbered species of both heparin/HS and DS that possess affinity, albeit weak, for NK1. Application of the GMSA technique in a newly developed selection mode has allowed us to make simultaneous assessments of the relative affinities for NK1 of differently sulfated oligosaccharides. The results clearly indicate that affinity is primarily influenced by sulfate density, even across different GAGs and ligand sizes and is not dependent upon the presence of sulfation at any specific ring position (consolidating previously suggestions from the behavior of differently sulfated GAG chains (
)). Importantly, NK1 does not discriminate between HS/heparin and DS dp4s of equivalent sulfate densities; affinity is, thus, essentially independent of hexosamine identity and glycosidic linkage within IdoA-containing GAGs.
A major, unresolved issue had been whether the dual GAG binding properties of NK1 and HGF/SF are due to the existence of two distinct, non-overlapping binding sites with exclusive GAG preferences or of a single binding site that accommodates both GAGs non-selectively. The former situation, although not common, occurs for example in TSG-6, where there are separate binding sites for hyaluronan and HS/heparin (
Mahoney D.J.
Forster M.J.
Blundell C.D.
Fries E.
Milner C.M.
Day A.J.
) and also in HARE, where hyaluronan/chondroitin sulfate and heparin bind independently to different sites (
Harris E.N.
Weigel P.H.
). Previously we showed that HS and DS could compete for binding to NK1 in a GMSA assay (
). However, in the case of TSG-6 it is known that binding of GAG to one site can indirectly influence GAG binding to the other site even though they are non-overlapping and cannot be occupied simultaneously (
Higman V.A.
Redfield C.
Noble M.E.M.
To further investigate the situation in NK1, we compared NMR titrations of [15N]NK1 with heparin and DS oligosaccharides. NMR provides residue-specific information and, by monitoring the protein rather than the GAG, thereby complements GMSA. The broadly similar cross-peak shifts elicited by the DS dp6 and heparin dp4 species indicate similar protein contacts and, thus, overlapping positioning, which is consistent with a single binding site accommodating either GAG. However, in the absence of the complete resonance assignments of the NH cross-peaks of NK1, it was mostly not possible to identify which specific amino acid residues were principally affected in these titrations. Nevertheless, using more limited sets of published data from NMR-monitored titrations of low molecular weight heparin and sucrose octasulfate with the N-domain and/or NK1 (
Zhou H.J.
Casas-Finet J.R.
Coats R.H.
Bottaro D.P.
Byrd R.A.
Biochemistry. 1999; 38: 14793-14802
), a few amino acid residues that were affected in both titrations, namely Asp-68, Cys-70, Arg-73, and Leu-80, could be tentatively assigned. It is notable that Arg-73 is implicated, as it had been previously identified as a pivotal residue in the NK1·heparin dp14 crystal complex (
The existence of a smaller cohort of NH cross-peaks that shift more strongly in the presence of DS dp6 than with heparin dp4, may signify subtle differences in the contacts made by different GAGs within the binding site. However, it may also reflect additional contacts made by the extra disaccharide unit present in the dp6. In this context it is notable that even though protein contacts are seen primarily between the N domain (Lys-58, Lys-60, Thr-61, Lys-62, Lys-63, Arg-73, and Gly-79) and a core dp4 segment of the dp14 in the crystal complex (
), a fifth monosaccharide (a GlcNS residue) contiguous with the non-reducing residue of this dp4 sequence can make an additional ionic contact with a further N-domain residue, Arg-76. In addition, some crystals also showed evidence of the interaction of a dp2 extension on the reducing side of the dp4 sequence, with a secondary cluster of basic residues (Lys-132, Arg-134, and Arg-181) on the face of the K1 domain. However, in the crystal this particular K1 domain belongs to the adjacent protomer in a head-to-tail NK1 dimer. Interestingly, the isolated K1 domain does exhibit independent heparin affinity in vitro, although it is significantly weaker than the isolated N domain (
). NMR data suggests, however, that NK1 in solution, unlike in the crystal, exists as a monomer.
D. Uhrin, unpublished data.
Thus, depending upon the interdomain configuration in solution, it is possible that oligosaccharides longer than dp4 could make further contacts in the N-domain outside the core binding site and additionally may bridge across to the contiguous K1-domain, thereby eliciting additional cross-peak movements in NMR.
Further useful comparisons may also be made between our observations and the contacts evident in the NK1-dp14 crystal structure. In the latter the IdoA situated at the non-reducing end of the core dp4 segment makes just one protein contact via a hydrogen bond between its 2-O-sulfate group and the key Arg-73 residue (that is also engaged in three other contacts elsewhere, one of which is with a 6-O-sulfate). It might, therefore, be expected that this 2-O-sulfated IdoA would not be essential for interaction, and indeed when such a residue was removed from the hexasulfated heparin dp4 to generate the pentasulfated dp3 it had only slightly reduced NK1 affinity. Also, the disulfated DS dp3 and dp4 species lack such 2-O sulfation but possess affinity.
It is also notable that two of the sulfates in the core dp4 segment (i.e. the central 2-O- and N-sulfates) make no observable contacts with NK1 in the co-crystal. This suggests that tetrasulfated dp4s specifically lacking these two sulfates should bind without compromising affinity. Although it is not possible to selectively remove these two specific sulfates, our GMSA analyses demonstrate that tetrasulfated dp4s continue to bind well irrespective of whether they lack both copies of the N-, 2-O-, or 6-O-sulfates. Furthermore, on the basis of the co-crystal it would be expected that a disulfated HS or DS dp4 would sustain a significant loss of specific contacts, which could explain the reduced affinities we observe. However, the equivalent affinities of the four disulfated dp4s with radically different sulfate patterns suggest that there may be sufficient conformational flexibility within the GAG sequence to allow alternative contacts to be made than those evident purely in this particular crystal complex. For example, the latter would suggest that interaction with a purely di-2-O-sulfated HS dp4 would be maintained by a salt bridge to only one of its two sulfates (at the non-reducing end), which seems unlikely.
A contributory factor in this flexibility may be the known conformational plasticity of IdoA (for reviews, see Refs.
Casu B.
Petitou M.
Provasoli M.
Sinaÿ P.
Trends Biochem. Sci. 1988; 13: 221-225
Glycobiology. 2000; 10: 1147-1156
), and indeed the presence of IdoA is primarily what distinguishes a binding DS from a non-binding chondroitin sulfate. In addition, it is known from FGF-heparin interactions that a kink in the overall helical structure of the chain can occur in certain trisaccharide segments, influenced in part by the conformation of the central IdoA, and such a kink can optimize protein contacts (
Raman R.
Venkataraman G.
Ernst S.
Sasisekharan V.
Sasisekharan R.
Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2357-2362
). The specific topology of the binding site in FGF2 also allows heparin to dock in either of its chain orientations (
). It is conceivable that similar structural accommodations may also operate to allow sequences of very variable sulfate density and patterning to make sufficient stable contacts with NK1 and HGF/SF. Consequently, it would be of great interest if future crystallography or NMR studies were to investigate how lesser sulfated sequences than fully sulfated heparin fragments are able to dock with NK1.
Overall, our observations with small oligosaccharides have conclusively revealed the operation of a single, high affinity but inherently low specificity GAG co-factor binding site in the NK1 region of HGF/SF. Affinity is dictated by the need for a minimum of an IdoA residue and two sulfates, which can be positioned in a variety of ways, located within a dp3/dp4 segment of either a HS/heparin or DS backbone. Separation of these minimal two sulfates by an intervening non-sulfated monosaccharide may be important, as an equally or higher sulfated dp2 does not bind NK1 (
). Thus, in vivo, initial interaction (and consequent co-factor activity) could be initiated almost anywhere within the IdoA-containing, sulfated domains of HS and DS, although highest activity will result from subsequent migration of the protein along the chain until it locates the most sulfated sequences of highest affinity. In HS these would correspond to the heparin-like cores of the sulfated domains, where N-, 2-O-, and 6-O-sulfates co-exist, whereas in DS they would be primarily where 2-O-sulfated IdoA residues are clustered.
The above characteristics of the GAG-binding site, as elucidated with minimal oligosaccharides, help to explain various disparate data in the literature relating to the binding of intact GAGs, suggesting that the behavior of small ligands does accurately reflect that of the natural polymers. The absence of any requirement for specific positional sulfation in HS/heparin mirrors what was previously observed in DS, where both mammalian and Ascidia nigra DS species bind and activate HGF/SF, although they possess either 4-O or 6-O sulfation of GalNAc, respectively (
). Also, the phenotype of the knock-out of the HS-2-O-sulfotransferase (the only HS sulfotransferase that exists as a single isozyme) (
Bullock S.L.
Fletcher J.M.
Beddington R.S.
Wilson V.A.
Genes Dev. 1998; 12: 1894-1906
) does not resemble that of either the HGF/SF or MET knock-outs (
Riethmacher D.
Isenmann S.
Aguzzi A.
Nature. 1995; 376: 768-771
Schmidt C.
Goedecke S.
Zschiesche W.
Sharpe M.
Uehara Y.
Minowa O.
Mori C.
Shiota K.
Kuno J.
Noda T.
Kitamura N.
), as would be expected if there was no specific requirement for 2-O sulfation. Indeed, HS derived from the knock-out animal continues to bind and activate HGF/SF similarly in vitro (
Merry C.L.R.
Swan D.C.
Backen A.C.
Beddington R.S.P.
). This may be a consequence of the increased N and 6-O sulfations of HS that occur in this knock-out and appear to compensate in overall charge for the loss of 2-O sulfation (
), thereby sustaining HGF/SF affinity and activity.
Conversely, the noted reduction in HGF/SF-mediated cell signaling, proliferation, and migration of carcinoma cells elicited by elevated expression of Sulf-1 (
Lai J.P.
Chien J.R.
Strome S.E.
Staub J.K.
Montoya D.P.
Greene E.L.
Smith D.I.
Roberts L.R.
Shridhar V.
Oncogene. 2004; 23: 1439-1447
Moser D.R.
Aderca I.
Matthews T.A.
Nagorney D.M.
Cunningham J.M.
Gastroenterology. 2004; 126: 231-248
), an extracellular HS 6-O-sulfatase, can be explained not by any specific requirement for 6-O sulfation but because 6-O-sulfates within the trisulfated, heparin-like disaccharides of HS are a major target of the Sulf enzymes (
Viviano B.L.
Paine-Saunders S.
Gasiunas N.
Gallagher J.
Saunders S.
Ai X.
Do A.-T.
Kusche-Gullberg M.
Lu K.
Emerson C.P.
). By reducing the overall sulfate density of these highest affinity sites, the effectiveness of the endogenous HS as a HGF/SF ligand and co-factor is likely to be diminished.
The activity of small oligosaccharides also has implications for the mechanism of MET activation by HGF/SF. Heparin binding to NK1 does not induce any conformational change in the latter (
), although it is reported to dimerize NK1 in solution (
Tolbert W.D.
Daugherty J.
Gao C.F.
Xie Q.
Miranti C.
Vande Woude G.
Xu H.E.
), consistent with the crystal complex (
). HGF/SF predominantly forms a 1:1 complex with the full-length MET ectodomain independently of GAGs (
); however, heparin dramatically enhances MET ectodomain dimerization in the presence of NK1 (
). A truncated MET ectodomain can also form dimeric 2:2 complexes with HGF/SF (
Sandin S.
Petoukhov M.V.
Finch J.
Öfverstedt L.-G.
Miguel R.N.
Skoglund U.
Svergun D.I.
) probably via the same NK1-NK1 dimer interface seen in the heparin-NK1 crystal (
). Thus GAG binding and resultant dimerization may drive the formation of an active, dimeric, 2:2 complex of HGF/SF-MET.
In contrast to the above, it is unlikely that an oligosaccharide as small as a dp3/dp4 will effect dimerization of NK1 or HGF/SF. Indeed, during the NMR titrations with small oligosaccharides described in this study, the NK1 appeared to remain monomeric.3 This concurs with our previous observation that a monomeric HGF/SF, which has been cross-linked to an oligosaccharide and is, thus, physically unable to form a dimer upon the same GAG molecule, is also active (
). This implies that activation may be possible through two distinct mechanisms, dependent upon the size of the GAG, which may be analogous to the situation recently described with FGF2 (
Goodger S.J.
Robinson C.J.
Murphy K.J.
Harmer N.J.
Pye D.A.
). Small oligosaccharides may promote a 1:1:1 complex of GAG·HGF/SF·MET that can then more readily dimerize to form an active 2:2:2 complex. The latter may, however, be less stable and effective in signaling than a 1:2:2 complex that can form upon longer GAG species.
A notable difference from the situation seen with FGF2 is in the apparent correlation between GAG structure and signaling. For FGF2, specific losses of sulfation within a heparin dp4/dp6 species may have little effect upon FGF2 binding, but can abolish activity, which was interpreted as possibly being due to a requirement for additional, receptor binding determinants within the oligosaccharide (
). With HGF/SF, activity appears to remain inextricably linked to GAG binding across a wide spectrum of sulfate densities and patterns. By comparison, this may suggest a lesser role, if any, for a putative GAG-MET interaction in the ternary complex, and indeed we have only previously detected a relatively weak intrinsic affinity of extracted, cellular MET for intact heparin (
In conclusion, a novel strategy of employing minimal oligosaccharides of variable, but well defined sulfation patterns coupled with new methodological developments in GMSA application has enabled us to refine the molecular parameters of GAG-HGF/SF interaction and MET activation. The data obtained should help in the future design of GAG mimics for HGF/SF inhibition. This approach should also have general applicability in elucidating the detailed recognition properties of many other GAG-binding proteins.
We are very grateful to Dr. Ermanno Gherardi (MRC Centre, Cambridge, UK) for the P. pastoris recombinant NK1 expression system and Prof. Jeffrey Esko (University of California, San Diego, CA) for the sulfated GAG-deficient CHO pgsA-745 cell line.
Download .pdf (.18 MB)
Received in revised form: December 24, 2008
Received: October 3, 2008
DOI: https://doi.org/10.1074/jbc.M807671200
© 2009 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.


text:
Las minas del rey Salomón es una película de acción y aventuras de 1985; la cuarta adaptación cinematográfica de la novela Las minas del rey Salomón de Henry Rider Haggard, aunque está muy pobremente adaptada. Dirigida por J. Lee Thompson y producida por Cannon Group, Inc. fue protagonizada por Richard Chamberlain, Sharon Stone, Herbert Lom y John Rhys-Davies. Filmada en las afueras de Harare, Zimbabue; esta cinta tenía un tono cómico y ligero que parodiaba a Indiana Jones.

Argumento
Jesse Huston, una hermosa joven, contrata los servicios del cazador Allan Quatermain para encontrar a su desaparecido padre, un famoso arqueólogo, que busca las legendarias minas del Rey Salomón. Descubren que ha sido secuestrado por alemanes al servicio del Coronel Bouckner, los cuales tienen como aliados al mercader árabe Dogati, porque descubrió el mapa verdadero que indica el lugar de las minas, las cuales ellos quieren obtener para poder enriquecerse a gran escala.

Una lucha por esas minas empieza con ello.

Reparto

Producción
Se hizo esta película, porque se quiso imitar así el éxito que habían tenido las películas de Indiana Jones.

Recepción
Aunque no les gustó a los críticos, esta película tuvo aun así suficiente éxito como para que se hiciese en 1987 una secuela de ella.

Véase también

Referencias

Enlaces externos

Películas de aventuras
Películas basadas en novelas
Películas ambientadas en Namibia

redpajama_set_name: RedPajamaWikipedia


need those last match stats at every match!
told me the guy that after every ettv/official he got, hes avi get in to crossfire site as buStEd.
so no-one is allowed to join that mgc ever again or what?
Ea$y 4 estonian superstar and amazing player Mr Cr!m!nal!


RITUAL INCENSE BURNER | Richard Bell for Psalt Design.
Incense has been used for thousands of years throughout ritualistic processes & is commonly used in homes to promote relaxation, positivity & creativity. The 'Ritual Series Incense Burner' blends tradition & minimalism to create an interior product for the design conscious individual. Each brass burner comes with a piece of Soladite, the stone of harmony, which has connections with the higher mind, confidence & balance. The aim of this is to act as a reminder to invest time into the mind, body & soul.
The 'Ritual Series incense burner' is manufactured from brass & references common manufacturing marks formed whilst bending metal. The piece is finished with a satin detail which is lacquered to prevent tarnishing.


text:
Q: Selection of interaction terms for logistic regression I'd like to use regularized logistic regression to classify elements (DNA sequences, i.e., character strings) into one of two categories based on the presence/absence (1/0) of many (k=50) features (DNA sequence motifs, i.e., substrings). I have approximately 20000 elements, which are split approximately 90:10 between my two classes.
I expect there to be significant interactions among some of these features (I mean non-additive effects between specific pairs of features), so I believe I want to include interaction terms in my model. Doing this exhaustively would mean examining a large number of interactions (50 choose 2=1225). To me, this seems bad--I don't think I have enough data to accurately fit a model with so many terms, and I would expect fitting such a model would be computationally challenging.
If I were working with a smaller number of features, would it be best to fit a model with all possible interactions, and then perform a post-hoc analysis of which terms significantly improve the model? For my current case, is it valid to pre-filter for specific interactions of interest (by identifying pairs of features that are enriched in (cooccur more often in) one class of elements vs. the other)? Thanks for your input.

redpajama_set_name: RedPajamaStackExchange


Fairfield Gardens Shopping Centre is located at 180 Fairfield Road, Fairfield and is easily accessible via a number of main roads.
Fairfield Gardens offers plenty of parking for your shopping convenience, with many spaces located under shade sails or cover. The one level layout ensures ease of accessibility regardless of where you park. A number of disabled parking and parents with prams parking facilities are also available near main entry ways, and free to eligible members of the public.
A Translink Bus Station is conveniently located just outside the Centre, next to 7Eleven at the Brougham Street entrance. For more information and to access bus timetables please contact Translink on 13 12 20 or visit the Translink website.
Fairfield Train Station is located approximately 250m directly behind the Centre. For information on timetables and routes, please contact Translink on 13 12 20 or visit the Translink website.
A Taxi Rank is located just down from BWS at the front of the Centre. Booking can be made by contacting Black and White cabs on 13 10 08 or Yellow Cabs on 13 19 24.


The Ableware 736030000 Universal Hand Clip by Maddak can be found in our Household Products product category within the Aids for Daily Living. Wholesale Point is an authorized dealer for Ableware by Maddak - so dig in, research, compare, ask questions and buy with confidence. We are pleased to offer Ableware by Maddak, Apex Carex, Briggs Healthcare, Essential Medical Supply, Forever New, Juvo, McKesson, Skil Care, Stand-A-Roo, Stander Inc, Tenura in a safe and secure online shopping experience plus free shipping within the continental United States when your order is at least $35. This particular item's manufacturer part number (MPN) is 736030000, the UPC code is 742645013501, has 1 year warranty and was made in USA.


text:
Musicians hating their own songs
Story behind Korn's Blind
Ben Stiller as James Hetfield
5FDP Spencer in quick-fire interview
By Christina O'Neill
Drummer Jeremy chooses Metallica over Slipknot and Breaking Bad over Game Of Thrones
Five Finger Death Punch sticksman Jeremy Spencer has taken part in a rapid-fire interview on his personal tastes.
He is asked 'either or' questions such as 'touring or recording,' 'Mortal Kombat or Street Fighter' and 'download or vinyl' in the short clip filmed by Impericon.
Asked to pick between the two bands, he chooses Metallica over Slipknot – after previously having cited Hetfield and co as inspiration.
The video comes after the band's latest album Got Your Six entered at no.2 in the Billboard 200 album chart – their third record to go straight to that spot after both volumes of 2013's The Wrong Side Of Heaven And The Righteous Side Of Hell.
The band made headlines last month after they cancelled their shows in France due to terror threats and security concerns after the Paris terrorist attacks.
Christina O'Neill
Former TeamRock news desk member Christina joined our team in late 2015, and although her time working on online rock news was fairly brief, she made a huge impact by contributing close to 1500 stories. Christina also interviewed artists including Deftones frontman Chino Moreno and worked at the Download festival. In late 2016, Christina left rock journalism to pursue a career in current affairs. In 2021, she was named Local Weekly Feature Writer of the Year at the Scottish Press Awards.
Marilyn Manson faces new lawsuit alleging sexual assault on a minor
"Iron Maiden fans are the coolest people on the planet": what your favourite band apparently says about you, according to this hilarious new video
Watch pro-shot footage of Babymetal's comeback shows in Japan this past weekend, which included two unreleased songs and a very interesting tease
Linkin Park are teasing something big to do with Meteora

redpajama_set_name: RedPajamaCommonCrawl


text:
United Express operation at DCA is less reliable?
Disclaimer: This post shall dive rather unabashedly into speculation and extrapolation from anecdotal evidence. In addition, the author harbours personal displeasure towards the "Express creep" into what until recently was an all-mainline United Airlines operation at DCA, the reasons being at least three-fold: 1) compromised service offerings of the regional operators v/v mainline service, e.g. a lack of live air traffic control audio (Channel 9), or any other audio options for that matter; reduced overhead bin space; reduced offerings in the forward cabin; 2) a perception of diminished reliability, as regionals operate with thinner scheduling buffers and reduced availability of spare (i.e. potential substitute) aircraft; and 3) a perception that reduced crew hiring requirements and comparatively Lilliputian pay negatively affect safety.
In checking the status of today's DCA-ORD flights on united.com, your fearlessly inquisitive author discovered a 55 minute departure delay on today's UA 7670, the scheduled 6am operation on DCA-ORD (which used to be UA 601 with mainline service...). In seeking to understand the operational implications, consider the following four facts: 1) yesterday evening's sole United Express (UAX) operation into DCA, UA 7669 (ORD-DCA, scheduled 7:05p-9:55p), was cancelled; 2) the aircraft operating UA 7669 makes a RON (remains overnight) at DCA and operates UA 7670 the following morning; 3) Shuttle America, operator of the UAX service at DCA (and owned by parent company Chautauqua Airlines), ferried an Embraer E70 aircraft this morning from CMH (Columbus, OH), which arrived at DCA at 6:08am and, by logical argument, must have provided the aircraft for this morning's delayed UA 7670; and 4) upon arriving at DCA on UA 600 earlier this week (at approximately 9am), I noticed that UA 7670 was cancelled (the E70 was still parked at a gate adjacent to that which my B757 would occupy), while the morning's mainline service to ORD all seemed to operate.
With apologies about the non- reader friendly format of the extended argumentation above, that is to say, about the decided lack of conventional punctuation, some closing points: 1) The above evidence is, admittedly, anecdotal and non-rigorous, particularly in regards to point 4, where I didn't even provide the superficial context that I at least afford to consideration of today's flight in points 1-3; 2) Cancelled flights and empty aircraft ferries are a normal occurrence in this industry; 3) That UA / UAX would ferry an E70 on CMH-DCA, instead of simply cancelling DCA-ORD and revenue-flying the aircraft on CMH-ORD, hints at concern about minimizing passenger disruption, which is laudable.
That said, the complexities of reliable United-branded operations on DCA-ORD and v/v would be minimized with all-mainline operation. To say nothing of my objections to outsourced "Express" operations a priori.
So go out, loyal constituents, and write the Customer Relations department at your preferred airline, demanding maximization of mainline service and minimization of the ironically-labeled "Express" variety! Don't pay any heed to how deceptively attractive the little bundle of aluminum, scrubbed all nice and clean, might look!
Scatter-brained, high-flying story-telling
Dear foolish readers, er, reader: (for you could be consuming something worth your time)
What follows is a true story, in the full spirit of the word 'true.' It not only truly happened, but the chronicle was truly written but minutes after the event, taking the form of an email that was instantly dispatched from the wireless device it was crafted upon, just as the wireless device encountered (naturally enough) a wireless signal, upon its carrier's reaching the terra firma of a runway at Chicago's O'Hare International Airport. I risk a lawsuit -- just as I had with the previous post; what lengths I go for my readership of no one! -- with what follows, as it was intended as privileged communication to the message's original recipient, not for posting upon every iPhone from Dakar to Dolny Slask, every LCD from Virgin America's seat-back variety to, well, the point is made.
Date: Unification day (no, not October 3, but the First of this month, when CO and UA, having said "I do," giddily stole away into the Red Carpet -clad Presidential Club suite for, well, I might have some youngin' readers here. No, of course I don't, who am I kidding.) Airbus even celebrated with a full-page advert in the Financial Times: (apologies for the poor readability; blame the iPhone camera)
Place: the District
Time: early afternoon
The deets --
Man oh man, today's airport arrival experience was among my most dramatic ever! Read on; I write from aboard UA 621, presently some 24 minutes into the DCA-ORD sector.
I arrived in Rosslyn at 1:30p as expected, and I was descending the long elevator shaft for the Metro moments thereafter, in possession of Peugeot road bike, Samsonite roll-aboard, and Briggs and Riley backpack. (The bike would serve as my get-away vehicle upon returning to DCA a few mornings later, thereby beating the gridlock automobile traffic on the roads and bridges into central DC.) And then the fun started.
Sure enough, the blue line departed just as I arrived on the platform via elevator, and the next would come only after the obligatory and interminable (and unacceptable) midday wait. The next DCA-bound subway rolled into Rosslyn around 1:50p.
Once on the Metro platform at DCA, a sprint to the elevator proceeded, followed by discovery of a new route to my usual Peugeot lock-up spot, adjacent to the US Airways hangar. I kissed the maddog (my term of endearment for the fine wheels) good-bye, and continued to run (and sweat - yes, it's unpleasant but true!) into the terminal and up to the terminus of a shockingly lengthy queue for priority security screening. The time was 2:15p. My scheduled departure time was 2:35p; hence, doors would close in ten minutes. The wait to get to the ID checker appeared to be a good 15 minutes. That would not do.
Eschewing all semblance of elegance, I got the attention of a non-TSA line attendant and pressed my case, underscoring that my qualifications for the premium lane were not only elite status, but F-class (achieved through upgrade) to boot. :) And it worked! I was escorted about two-thirds of the way forward, and I furthermore "pardoned" my way past another 10% of the line. My wait thereby reduced to three or four minutes, I was through the checkpoint by 2:21. What good fortune!
I decided to double my bet. I could have easily walked to the gate - I was in possession of a luxurious quantity of 4 whole minutes, after all! - but decided to hit the restroom and change into some fresh clothing. Bear in mind that, at this time (and pardon the directness), sweat was beading down my neck, and I was feeling positively disgusting. No sooner than I'd entered the restroom, I heard "final boarding call, United 621!". But it was only 2:22, and I was determined to take full advantage of my remaining 180 seconds.
I finished my sartorial task most expeditiously and emerged into the male-female-combined world at 2:23p. I was at Gate 31 while my phone still displayed 2:23 (it would tick to 2:24 a few seconds later). The door to the jetbridge was open, the jetbridge was still attached to the New Colours A319, but there were no agents in sight! Finally, just over a minute later, after waiting patiently (what else could I do?!), I saw the jetbridge begin to detach. And an agent made an appearance. "Excuse me," in said. No response, not even a turn of the agent's head or eyeballs, despite being about two feet away from me. "Excuse me," I tried again, and still no acknowledgement. I could already see the angry email to United's Customer Relations that I would subsequently compose. But then, unexpectedly, a helpful query: "oh, are you on this flight?" Why, yes. Why else would I be standing at the jetway? I show my boarding pass and the demeanor improves further. I'm invited down the jetway in no time.
Now firmly ensconced within the aluminum skeleton of the jetliner, one further adventure awaited that momentous half-hour. I saw someone occupying my 2D. And I was told to gate-check my Samsonite. I do not admit this with pride, but the seasoned traveller within suddenly made a full-on appearance. I stubbornly began checking all the overhead bins in F, and finding only meager space for my backpack and maddogging helmet, proceeded to investigate bins in the forward part of Economy Plus. I was determined, even though the gate agent was already in the process of writing out a gate-check tag and affixing it to my bag, and suddenly - yes, some elusive space! Again with some dissatisfaction, I admit to having forcefully and clumsily rearranged the contents of that overhead bin and then expertly shoving my over-sized Samsonite into the created space. And I then took 1D, without so much as an evil-eye to the occupant of *MY* 2D! :)
Fondly from flight, now 0:56 past wheels up, and above western Ohio, (though, obviously, my note will be dispatched only upon making contact with one of ORD's fine runways)
Awaiting a barrage of criticism from non-existent readership, pertaining to all the explicit and tacit haughtiness that, it might be imagined, oozed forth from me...
Post script: For the sake of posterity, here's what the blurry text in the Airbus advert above really says: Read Ad Astra It's the Best Read Ad Astra (et cetera). Ok, here's what it really says:
Congratulations to everyone at Continental and United on the merger creating the new United, a truly world-class global airline. Your partners at Airbus, the world's leading aircraft manufacturer, wish you all the best. October 1, 2010 was a big day for the world of aviation, and for everyone that wants to fly.
07/10: Big-picture musings
I will not commence this post with a renewed vows of frequent and substantive posting. I'll let the following (true) statistic speak of the volume of readers that have been begging, badgering, and downright being belligerent about the dearth of content: Google currently owes me less than the typical price of a cup o' Joe in advertising fees for the past few months. Hopefully that statement is sufficiently vague to dodge those highly-paid Silicon Valley lawyers, who are just itching to sue the latest dirt-poor blogger for untold damages at having revealed their precise remuneration from Google Adwords. Based on my earnings, I can't even afford the Metro fare to the courtroom, to say nothing of needing to hitchhike my way back.
Those pleasantries aside, I have navigated a transition into new professional endeavors over the past few weeks, and the upheaval having passed, I trust that there's at least somewhat of a chance for sustained value generation on these pages over the weeks ahead. And the impetus for the vagueness is an omnipotent, oddly mysterious, 'just because', its being one of the few perks (ok, the only one) of being Founder, Chairman and CEO of Ad Astra Publishing and Pontificating, NLC (like an LLC, but better, with no liability whatsoever!) And yes, dear reader, the way this writing is flowing, I too identify this post as being at risk of speedy revision.
In just-breaking news, markets have not stood still since I last scratched the virtual chalkboard that is this blog. September spawned a sustained and, so far, unbroken move towards the upside, with the broad-market /ES contract now looking over-extended, though not necessarily without potential for one last pop, before the inescapable tendency towards mean-reversion says hello. In any event, 1175 looms as important forthcoming horizontal resistance, as do 1200 and 1210 further up the chart.
A 180d/4h /ES chart follows. From here on, such notation will convey the following: first, the time period of the chart (here, 180 days); second, the time period of each bar (here, 4 hours). /ES is, of course, a 24/5 contract (meaning that it trades for approximately 120 hours per week); hence, a 180-day chart of /ES will contain far more data that, say, a 180-d depiction of SPY, its corresponding exchange-traded fund, which trades for but 32.5 hours per half-fortnight. And I trust, reader, that you'll somehow forgive the quality of this late hour's missive, and return for more dreary punishment tomorrow. Yes, I did say tomorrow.


INSTRUCTIONS FOR THE ANNUAL FINANCIAL REPORT FORM This form is due to our Office on the 15th day of the 5th month after the end of your fiscal year. (Organizations may submit an IRS Form 990 or 990-EZ to this Office instead.) To complete this form, first fill out the General Information section of the form. Next, complete all schedules relevant to your organization. Start with Schedule 5, and finish with the Financial Summary. Note that Schedule 1 and Activity Statements are required of all organizations. When completed, the form should be signed and dated by the Chief Executive Officer and Chief Financial Officer of the organization. There is no fee for filing this form. You may mail, overnight or fax the form to us. General Information � � � Employer's Identification Number (EIN) � Identification number assigned to a nonprofit by the Internal Revenue Service. "Fiscal Year Beginning" and "Fiscal Year Ending" � Organizations establish the dates of their fiscal year. If an organization wishes to change its fiscal year, it should contact our Office. Charity Registration Number - Number assigned to your organization by the Public Charities Division.
Schedule 5: Special Events and Fundraising Sales Use this schedule for special events like bingo games, golf tournaments, dinners, auctions and for sales of products like candy, fruit, t-shirts and concessions. Schedule 4: Contracts with Commercial Co-Venturers (CCV) Commercial Co-Venture (CCV) - any agreement between a business and a charity in which the business advertises that the sale of its goods or services will benefit a charitable organization and the price of the good or service does not exceed that normally charged. An example of a CCV -- a restaurant might advertise that for every hamburger sold, it will donate 25 cents to a particular charity. The charity should report on Schedule 1, Line 3 only its income from the event. Schedule 3: Contracts with Professional Fundraising Counsels (PFRC) Professional Fundraising Counsels (PFRC) - any individual or business which contracts with a charitable organization to plan, manage or prepare material for a fundraising campaign which the charitable organization will conduct. A Professional Fundraising Counsel, however, does NOT solicit funds. A PFRC might prepare grant proposals or plan a mailing for charitable organizations. Schedule 2: Contracts with Professional Fundraising Solicitors (PFRS) Professional Fundraising Solicitor (PFRS) - an individual or business which contracts with a charitable organization to solicit contributions for it. For example, a PFRS might call citizens or go door-to-door to ask for contributions. Bingo operators are considered professional solicitors under state law but not federal law. Information on bingo promoters should be entered on Schedule 5, Special Events and not Schedule 2. Thus, a charity which has hired a professional solicitor to conduct a special event would complete the Annual Financial Report Form in the same way as IRS Form 990 or 990-EZ. Schedule 1: Contributions Line 3 Line 5 Line 10 . Line 12 "Commercial Co-Venture" � See definition under Schedule 4 above. "Special Events and Fundraising Sales" � See definition under Schedule 5 above. "Other" � In this section, you may list other kinds of solicitation and the proceeds from them Federated Fundraising Agencies - a group of independent charitable organizations which have voluntarily joined together to raise and distribute contributions (i.e. United Way).
Activity Statements Required of all Organizations Financial Summary Line 1 "Direct Public Support" � donations received from direct mail, telephone solicitations, commercial coventures, door-to-door solicitations, special events, telethons, and sales of goods and services to raise money for charitable purposes. Donations to be entered on this line may be cash, securities, or property of marketable value. It does NOT include donated services or use of facilities. It includes membership dues if there is NO qualification for membership, i.e. Friends of the Library. "Indirect Public Support" - monies received from other charitable organizations, affiliates and federated fundraising agencies (for example, United Way). "Government Grants" - monies received from, and by application to, federal, state, or local governments. "Program Service Revenue" - monies your organization receives for providing services it was created to offer and for which it may have received tax exempt status. (Examples include admission fees to performances and registration fees for conferences. Include membership dues if there is a qualification for membership.) "Other Revenue" - monies from other sources, such as interest or dividends earned. It also includes local government allocations to volunteer fire departments. "Program Services" - monies which your organization spent directly on goods and services for its charitable programs and purposes. This does NOT include fundraising or administrative expenses, or combined fundraising/public information expenses. "Fundraising" � costs of soliciting donations. Include expenses in telemarketing, mailing, advertising, applying for grants, and fees of professional solicitors or counsels, except if they are involved solely with special events such as a bingo game. In those cases, use only Schedule 5, Special Events. "Management and General" - administrative costs for running the organization. Examples of such costs are accounting fees, bank charges, costs of board meetings and board insurance, post office box rent, office supplies, some or all of the salary of the executive director and his/her administrative assistants. "Fund Balances or Net Worth at the beginning of the year" � all of your organization's assets minus any liabilities at the beginning of the fiscal year. Such assets would include cash, certificates of deposits, stock, real estate, major equipment (like vehicles), etc. This figure should match the ending balance of your previous report, if any, filed with this Office. "Fund Balances or Net Worth at the end of the year" � all of your organization's assets minus any liabilities at the end of the fiscal year. Such assets would include cash, certificates of deposits, stock, real estate, major equipment (like vehicles), etc. Line 17 MUST EQUAL equal Line 20. "Assets" (as of Fiscal Year End) - Items of value owned by the organization. Assets include cash, certificates of deposits, stock, real estate, major equipment (like vehicles), etc. "Liabilities" (as of Fiscal Year End) � Debts owed by the organization. "Fund Balance" � Net Worth at the end of the fiscal year. This figure MUST EQUAL Line 17.
Both the chief executive officer and chief financial officer of your organization must sign the Annual Financial Report on its last page. If one person serves as both officers, then that person must sign in both places.
This form, including any attachments, is a public record and a copy will be provided upon request to any interested person. Instructions for completing the form are attached at the end of the form. There is NO FEE for filing this form.
If your organization employed a professional solicitor during this fiscal year should you complete this schedule. (However, if the solicitor helped only with a special event like bingo, do NOT use this schedule. Use Schedule 5, instead) If insufficient room in the form below, copy this form and attach sheet.
4. Total public donations* 5. All Payments to PFRS 6. All other fundraising expenses of the organization for each event, sale or campaign 7. Total Expenses (Line 5 plus Line 6)** 8. Net proceeds (Line 4 minus Line 7) * On Line 4, do NOT exclude monies paid to or retained by PFRS (i.e. monies reported on Line 5). All monies listed on Line 4 must be included on Schedule 1 under the section Direct Public Support. ** Total from Line 7 in this schedule must be included on Financial Summary, Line 10 or 11.
If your organization employed a professional counsel during this fiscal year, complete this schedule. (If the counsel helped only with a special event or fundraising sale, do NOT use this schedule. Use Schedule 5, instead.) If insufficient room in the form below, copy this form and attach sheet.
* From Line 4 above, include total of all payments to PFRCs on the Financial Summary, Line 10 or 11.
TURN PAGE -- CHIEF EXECUTIVE AND CHIEF FINANCIAL OFFICERS MUST SIGN THE BACK OF THE ANNUAL FINANCIAL REPORT FORM.
Please see instructions attached. If insufficient room in the form below, copy this form and attach sheet.
* Transfer net proceeds to the charity for all events from Line 6 above to Schedule 1, Line 3.
Direct Expenses mean the cost of any food, beverage, entertainment, rent and maintenance of building involved in a special event or the cost of any product or service sold. Also, include in Line 4 any taxes and any fees paid to professional fundraising counsels or solicitors who assisted with the sale or event such as a bingo game. ** Transfer Adjusted Gross to Schedule 1, Line 5, "Special Events."
As required by Section 33-56-30 of the Solicitation of Charitable Funds Act, this form shall be signed by the Chief Executive Officer and the Chief Financial Officer of the charitable organization. (If one person serves as both CEO and CFO, he or she should sign in both places below.) WE CERTIFY THAT THE INFORMATION FURNISHED IN THIS STATEMENT IS TRUE AND CORRECT TO THE BEST OF OUR KNOWLEDGE AND BELIEF.


Straight from the Aloha State, Sean Yoro is an Hawaiian grown artist who not only creates amazing murals but he does it while standing on a surfboard. All murals portray women swimming or bathing and they seem amazingly alive due to the reflection they have in real water.
Hula now resides in New York City and he picks abandoned places with natural water on the compound, takes his paint, a surfboard and transform it into a beautiful canvas. His approach towards murals got the attention of many street artists and of the entire community. His Hawaiian touch on creating art is fun and difficult at the same time. Painting from water is a challenge since you don't have below a steady ground. With practice and a natural born talent, his work is simply amazing.
All images are made in oil paint and due to the circumstances in which they are produces, it might take up to four days to finish a simple work. All portraits are large, hyperrealistic images of beautiful women partially submerged in water.
He keeps an eye open to find new places to draw his murals, he even applied to get permission to create some of his work on bridges. Until he gets it he keep transforming dilapidated constructions into incredible exhibitions. All his art can be admired on his website, created under his stage name Hula. There you can view all his latest work as well as buy some of his art on print. Be fast, his works are selling fast.


Joe Gianni started his banking career in the 1980s and has become a well-known leader in Connecticut. He'll play a more prominent role in the state as he was named Bank of America's president for the greater Hartford market at the start of 2018.
As market president for Hartford, Gianni will continue to work across the region to connect businesses, families and individuals to Bank of America's banking and investment teams. He will also lead the effort to direct the bank's resources in the market and the region to address local priorities to help build strong communities.
We have a large company with a lot of moving parts, so my job is to facilitate the relationships and provide access to the talent we have in this organization.
In addition to his role as market president, Gianni serves on the government relations team, where he engages with public officials and local leaders on policy priorities. Previously, Gianni served as the Northeast region executive, overseeing local market activity throughout the Northeast. He has been with Bank of America and its predecessor banking institutions in Hartford since 1989.
He succeeds Kevin J. Cunningham, who served as the bank's market president for Connecticut and, later, for greater Hartford when the role was divided between Hartford and Fairfield County.
Gianni serves as the executive sponsor of the local Domestic Violence Task Force employee network, helping to raise awareness and educate teammates about the issue. He is also an active leader in the community, serving on the boards of Foodshare, the Bushnell Center for the Performing Arts, the MetroHartford Alliance and Kingswood-Oxford School.


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Fimdefelice
FIM was established in 1976 by Stephen L. DeFelice, M.D. who coined the term nutraceutical. FIM is the leading advocate for encouraging natural substance medical research.
NREA
A Comparison of the U.S., European & Japanese Nutraceutical Health and Medical Claim Rules
Proceedings of this conference were published in RegulatoryAffairs, Vol. 5, Summer 1993
A COMPARISON OF THE U.S., EUROPEAN AND JAPANESE NUTRACEUTICALHEALTH AND MEDICAL CLAIM RULES
Discussions and Recommendations for the U.S.
A nutraceutical is any substance that can be considereda food or part of a food and provides medical or health benefits,including the prevention and treatment of disease. Such productsrange from traditional foods, isolated nutrients, dietary supplementsand diets to genetically engineered "designer" foods,herbal products and processed foods such as cereals, soups andbeverages.
The Foundation coined the term nutraceutical to [a) give anidentiy to a new health sector that offers enormous health andmedical benefits to the consumer, and (b) to encourage the creationof a new regulatory category. This conference will address theU.S., European and Japanese nutraceutical systems and will considerrecommendations by both the panel and the audience regarding waysto encourage the establishment of a much needed vigorous researchand development oriented nutraceutical system in the United States.
The Foundation will present its own recommendations regardinga new nutraceutical regulatory category as an addition to existingones, where a sponsor can, at costs generally substantially lowerthan those associated with pharmaceuticals and food additives,obtain an exclusive health or medical claim. The rationale isbased on the economic principles which led to the highly successfulOrphan Drug Act.
As part of the program, the case method will be used regarding designer foods, plant and animal dietary supplements, processed foods – cereal and a food additive. Audience input will be emphasized. A summary of the conference, including the recommendations for a new regulatory approach, will be sent to appropriate parties including attendees.
Lyn CioccaThe Foundation for Innovation in Medicine, Cranford,NJ
William J. CurranJ.D., LL.M., S.M.HYG., Frances GlessnerLee Professor of Legal Medicine, Harvard Medical School, Boston,MA
Stephen L. DeFeliceM.D., Chairman, The Foundation forInnovation in Medicine, Cranford, NJ
Victor L. FulgoniIII, Ph.D., Vice President, Nutrition,Kellogg Company, Battle Creek, Ml
Tadayasu FurukawaPh.D., President, Nutri-Quest, Inc.,St. Louis, MO
Nicholas HallPresident, Nicholas Hall & Co., Essex,United Kingdom
Paul A. LachancePh.D., Professor & Chairman, RutgersUniversity, Food Science Department, New Brunswick, NJ
Louis LasagnaM.D., Dean, Sackler School of Graduate BiomedicalScience, Tufts University, Boston, MA
Stephen H. McNamaraEsq., partner, Hyman, Phelps &McNamara, P. C., Washington, DC
Abbey S. MeyersExecutive Director, National Organizationfor Rare Disorders, New Fairfield, CT
Herbert F. PiersonJr., M.S. Ph.D., Pharmacognocist/ChiefConsultant, Preventive Nutrition Consultants, Inc., Woodinville,WA
Gerard PilleyPh.D., Member of the French National Councilfor Pharmay, Paris, France
Richard S. RivlinM.D., Chief Nutrition Service, MemorialSloan-Kettering Cancer Center; Professor of Medicine and ChiefNutrition Division, Cornell Medical Center, New York, NY
Hercules ASegalas, Managing Director, Paine Webber Inc.,New York, NY
Michael D. YoungM.D., Ph.D., Corporate Director, Regulatoryand Clinical Development Worldwide, The Procter & Gamble Company,Cincinatti, OH
PO Box 1220, Mountside, NJ 07092
fimdefelice@aol.com
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