Cistromic and genetic evidence that the vitamin D receptor mediates susceptibility to latitude-dependent autoimmune diseases.

The vitamin D receptor (VDR) is a ligand-activated transcription factor that regulates gene expression in many cell types, including immune cells. It requires binding of 1,25 dihydroxy vitamin D3 (1,25D3) for activation. Many autoimmune diseases show latitude-dependent prevalence and/or association with vitamin D deficiency, and vitamin D supplementation is commonly used in their clinical management. 1,25D3 is regulated by genes associated with the risk of autoimmune diseases and predominantly expressed in myeloid cells. We determined the VDR cistrome in monocytes and monocyte-derived inflammatory (DC1) and tolerogenic dendritic cells (DC2). VDR motifs were highly overrepresented in ChIP-Seq peaks in stimulated monocyte (40%), DC1 (21%) and DC2 (47%), P

Development and application of a sensitive high performance ion-exchange chromatography method for the simultaneous measurement of dopamine, 5-hydroxytryptamine and norepinephrine in microdialysates from the rat brain.

A high performance liquid chromatography (HPLC) method based on cation exchange separation has been developed for the measurement of dopamine (DA), 5-hydroxytryptamine (5-HT) and norepinephrine (NE) in microdialysates. The separation conditions have been optimised for using electrochemical detection. All three bioamines were resolved in less than 22 min using isocratic conditions. The optimum oxidation potential for the three bioamines was found to be +0.4 V vs. in situ Ag/AgCl reference electrode. Linear regression analysis of HPLC-peak area as a function of concentrations in the range 1-50 ng x ml(-1) gave coefficients of correlation between 0.998 and 0.999. The limit of detection for DA, 5-HT and NE was found to be between 50 and 100 pg x ml(-1) with a signal to noise ratio of 3:1. The method has been applied to the simultaneous measurement of the three monoamines in microdialysates from the medial prefrontal cortex under basal conditions and following the administration of the antipsychotic drug clozapine (10 mg x kg(-1) s.c.).

In vivo characterization of basal amino acid levels in subregions of the rat nucleus accumbens: effect of a dopamine D(3)/D(2) agonist.

Recent evidence demonstrates that two subdivisions of the nucleus accumbens, the dorsolateral core and the ventromedial shell can be distinguished by morphological, immunohistochemical and chemoarchitectural differences. In the present study, we measured basal levels of amino acids in microdialysates from both the shell and core subterritories of the nucleus accumbens in freely moving rats using HPLC with fluorescence detection. The effect of the dopamine D(3)/D(2) receptor agonist quinelorane (30 microg/kg s.c.) was then investigated in both subregions. With the exception of glutamate, histidine, and serine, which showed similar levels in both subterritories, alanine, arginine, aspartate, gamma-aminobutyric acid, glutamine, and tyrosine were significantly higher in the shell compared with the core. In contrast, taurine levels were significantly lower in the shell than in the core. A particularly striking difference across subregions of the nucleus accumbens was observed for basal GABA levels with a shell/core ratio of 18.5. Among all the amino acids investigated in the present study, quinelorane selectively decreased dialysate GABA levels in the core subregion of the nucleus accumbens. The results of the present study point to specific profiles of both shell and core in terms of: (1) basal chemical neuroanatomical markers for amino acids; and (2) GABAergic response to the DA D(3)/D(2) agonist quinelorane.

Solution structure of the third immunoglobulin domain of the neural cell adhesion molecule N-CAM: can solution studies define the mechanism of homophilic binding?

Homophilic binding of the neural cell adhesion molecule (N-CAM) mediates the calcium-independent cell-cell adhesion that is involved in neuronal development. Two hypotheses have been advanced for the mechanism of homophilic binding. Cell-based experiments have implicated each of the five extracellular immunoglobulin (Ig) domains of N-CAM in the homophilic adhesion interaction, and have predicted that the third domain (Ig III) self-associates. The alternative hypothesis is based on solution observations, which implicate a specific antiparallel interaction between the first two Ig domains (Ig I and Ig II). In order to test these hypotheses, we have determined a high-resolution solution structure of recombinant Ig III (sequence derived from chicken N-CAM) and examined the aggregation behavior of isolated Ig domains in solution. The structure shows that Ig III adopts a canonical Ig fold, in which the beta strands ABED and A'GFCC' form two beta sheets that are linked by a disulfide bond. In contrast to the demonstrated aggregation of Ig III on solid supports, we were unable to demonstrate self-association of Ig III under any of a variety of solution conditions. The structure shows that the surface of Ig III is dominated by two large acidic patches, which may explain our failure to observe self-association in solution. To evaluate the involvement of the Ig I-Ig II interaction in cell-cell adhesion, we designed a point mutation in Ig I (F19S) that proved sufficient to abrogate the Ig I-Ig II interaction seen in solution. However, the introduction of this mutation into full-length N-CAM expressed in COS-7 cells failed to affect N-CAM-mediated cell-cell adhesion. The inability to observe Ig III self-association in solution, combined with the failure of the F19S mutation to affect N-CAM-mediated cell-cell adhesion, suggests that, although solution studies can give important insights into the structures of individual domains, the interactions observed in solution between the domains may not be representative of the interactions that occur on the cell surface.

NMR structure of a concatemer of the first and second ligand-binding modules of the human low-density lipoprotein receptor.

The ligand-binding domain of the human low-density lipoprotein receptor consists of seven modules, each of 40-45 residues. In the presence of calcium, these modules adopt a common polypeptide fold with three conserved disulfide bonds. A concatemer of the first and second modules (LB(1-2)) folds efficiently in the presence of calcium ions, forming the same disulfide connectivities as in the isolated modules. The three-dimensional structure of LB(1-2) has now been solved using two-dimensional 1H NMR spectroscopy and restrained molecular dynamics calculations. No intermodule nuclear Overhauser effects were observed, indicating the absence of persistent interaction between them. The near random-coil NH and H alpha chemical shifts and the low phi and psi angle order parameters of the four-residue linker suggest that it has considerable flexibility. The family of LB(1-2) structures superimposed well over LB1 or LB2, but not over both modules simultaneously. LB1 and LB2 have a similar pattern of calcium ligands, but the orientations of the indole rings of the tryptophan residues W23 and W66 differ, with the latter limiting solvent access to the calcium ion. From these studies, it appears that although most of the modules in the ligand-binding region of the receptor are joined by short segments, these linkers may impart considerable flexibility on this region.

Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa 1. Isolation, characterization and chemical synthesis.

A novel conotoxin belonging to the 'four-loop' structural class has been isolated from the venom of the piscivorous cone snail Conus tulipa. It was identified using a chemical-directed strategy based largely on mass spectrometric techniques. The new toxin, conotoxin TVIIA, consists of 30 amino-acid residues and contains three disulfide bonds. The amino-acid sequence was determined by Edman analysis as SCSGRDSRCOOVCCMGLMCSRGKCVSIYGE where O = 4-transL-hydroxyproline. Two under-hydroxylated analogues, [Pro10]TVIIA and [Pro10,11]TVIIA, were also identified in the venom of C. tulipa. The sequences of TVIIA and [Pro10]TVIIA were further verified by chemical synthesis and coelution studies with native material. Conotoxin TVIIA has a six cysteine/four-loop structural framework common to many peptides from Conus venoms including the omega-, delta- and kappa-conotoxins. However, TVIIA displays little sequence homology with these well-characterized pharmacological classes of peptides, but displays striking sequence homology with conotoxin GS, a peptide from Conus geographus that blocks skeletal muscle sodium channels. These new toxins and GS share several biochemical features and represent a distinct subgroup of the four-loop conotoxins.

Association between the first two immunoglobulin-like domains of the neural cell adhesion molecule N-CAM.

The extracellular domain of N-CAM contains five immunoglobulin-like (Ig) and two fibronectin type III-like domains and facilitates cell-cell binding through multiple, weak interdomain interactions. NMR spectroscopy indicated that the two N-terminal Ig-like domains from chicken N-CAM (Ig I and Ig II) interact with millimolar affinity. Physico-chemical studies show that this interaction is significantly amplified when the domains are covalently linked, consistent with an antiparallel domain arrangement. The binding of the two individual domains and the dimerization of the concatenated protein were essentially independent of salt, up to a concentration of 200 mM. The residues in Ig I involved in the interaction map to the BED strands of the beta sandwich, and delineate a largely hydrophobic patch.

Folding, calcium binding, and structural characterization of a concatemer of the first and second ligand-binding modules of the low-density lipoprotein receptor.

The ligand-binding domain of the low-density lipoprotein (LDL) receptor is comprised of seven tandemly repeated ligand-binding modules, each being approximately 40 amino acids long and containing six conserved cysteine residues. We have expressed and characterized a concatemer of the first two modules (LB1 and LB2) of the human LDL receptor. Oxidative folding of the recombinant concatemer (rLB1-2), in the presence of calcium ions, gave a single dominant isomer with six disulfide bonds. Peptic cleavage of the short linker region that connects the last cysteine residue of LB1 and the first cysteine residue of LB2 yielded two discrete fragments, thus excluding the presence of intermodule disulfide bonds. The N-terminal module, LB1, reacted with a conformation-specific monoclonal antibody (IgG-C7) made to LB1 in the native LDL receptor. From this, we concluded that the first module was correctly folded, with the same set of disulfide bonds as LB1 of the LDL receptor. The disulfide bond connections of LB2 were identified from mass spectral analysis of fragments formed by digestion of the C-terminal peptic fragment with elastase. These data showed that the disulfide bonds of LB2 connected Cys(I) and Cys(III), Cys(II) and Cys(V), and Cys(IV) and Cys(VI). This pattern is identical to that found for recombinant LB1 and LB2. The concatemer has two high-affinity calcium-binding sites, one per module. An analysis of the secondary chemical shifts of Calpha protons shows that the conformations of LB1 and LB2 in the concatemer are very similar to those of the individual modules, with no evidence for strong interactions between the two modules.

Calcium is essential for the structural integrity of the cysteine-rich, ligand-binding repeat of the low-density lipoprotein receptor.

Seven cysteine-rich repeats form the ligand-binding region of the low-density lipoprotein (LDL) receptor. Each of these repeats is assumed to bind a calcium ion, which is needed for association of the receptor with its ligands, LDL and beta-VLDL. The effects of metal ions on the folding of the reduced N-terminal cysteine-rich repeat have been examined by using reverse-phase high-performance liquid chromatography to follow the formation of fully oxidized isomers with different disulfide connectivities. In the absence of calcium many of the 15 possible isomers formed on oxidation, whereas in its presence the predominant product at equilibrium had the native disulfide bond connectivities. Other metals were far less effective at directing disulfide bond formation: Mn2+ partly mimicked the action of Ca2+, but Ba2+, Sr2+, and Mg2+ had little effect. This metal-ion specificity was also observed in two-dimensional 1H NMR spectral studies; only Ca2+ induced the native three-dimensional fold. The two paramagnetic ions, Gd3+ and Mn2+, and Cd2+ did not promote adoption of a well-defined structure, and the two paramagnetic ions did not displace calcium ions. The location of calcium ion binding sites in the repeat was also explored by NMR spectroscopy. The absence of chemical shift changes for the side chain proton resonances of Asp26, Asp36, and Glu37 from pH 3.9 to 6.8 in the presence of calcium ions and their proximal location in the NMR structures implicated these side chains as calcium ligands. Deuterium exchange NMR experiments also revealed a network of hydrogen bonds that stabilizes the putative calcium-binding loop.

1H NMR studies of sarafotoxin SRTb, a nonselective endothelin receptor agonist, and IRL 1620, an ETB receptor-specific agonist.

1H NMR studies on the nonselective endothelin receptor agonist sarafotoxin SRTb have identified a helix between residues Asp 8 and His 16, and a beta-turn involving residues Cys 3 to Met 6; however, the biologically important C-terminal five residues were found to be conformationally variable. The average RMSD, measured for the final 43 refined structures to the average structure over residues 1-16, was 0.78 +/- 0.18 A for the backbone atoms and 1.39 +/- 0.22 A for all atoms. The torsion angles Cys 3 psi/Lys 4 theta, Thr 7 psi/Asp 8 theta and Gln 17 theta were identified as sites of conformational variability. Differences were found between the structures in the bicyclic loop region for SRTb and those published for ET1, another nonselective receptor agonist, which may explain the observed differences in potency of these peptides. The conformation of an ETB receptor-specific agonist, IRL 1620, which lacks the N-terminal seven residues and the two intrachain disulfides, was found by NMR and circular dichroism spectroscopy to be predominantly random coil, despite the fact that its affinity for the ETB receptor almost equals that of ET1. However, close analysis of the NMR results indicated the presence of turn-like structures, or a nascent helix, in the part of the sequence corresponding to the helical region in the parent peptides. These results suggest that the helical conformation may be required for ligand binding to the ETB receptor as well as to the ETA receptor.

Conformational stability of the endothelin/sarafotoxin family of peptides.

There are significant differences between the structures reported for members of the endothelin/sarafotoxin family of peptides, but also for the same peptides studied by different groups, raising the possibility that some of the differences are attributable to variation in solution conditions rather than intrinsic structural heterogeneity. We have shown, using circular dichroism spectroscopy and equilibrium sedimentation, that the secondary structures of these peptides are little affected by wide variations in pH, or by self-association. Although acetonitrile has a pronounced effect on the extent of peptide self-association it does not appear to alter the backbone structure of sarafotoxin SRTb, and has only minor effects on endothelin-1 and endothelin-3. The observed conformational variation thus appears largely to reflect sequence-dependent differences.

Solution structure of the toxic octapeptide, lophyrotomin.

Lophyrotomin is a toxic octapeptide, first isolated from larvae of the sawfly Lophyrotoma interrupta, which causes the death of cattle and sheep. It appears to act principally on the liver, however very little is known about the cellular site and mechanism of action. In the present study lophyrotomin was synthesized using solid-phase peptide synthesis, and the structure examined with two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Two-dimensional correlation experiments (COSY and TOCSY) enabled the assignment of many of the resonances. Conventional NOESY experiments did not produce inter-residue information, however the alternative rotating frame NOE experiment (ROESY) resulted in intra-residue alpha N, and sequential alpha N and NN NOEs, permitting the sequence-specific assignment of all resonances. The presence of few additional shortage NOEs and the absence of any long-range NOEs in the ROESY spectra indicated a lack of persistent secondary structure. The results from circular dichroism (CD) spectroscopy experiments were consistent with the NOE data, as addition of high concentrations of the denaturant urea produced no changes in the lophyrotomin CD spectrum. This conclusion was further supported by 13C spin-lattice relaxation studies, which indicated that the peptide is a flexible molecule, by examination of the alpha-carbon chemical shifts, and by amide proton exchange rate measurements. Consequently it appears that if this peptide has to adopt a well defined structure to exert its biological activity, it must do so on interaction with other molecules, such as a receptor.

Conformation of sarafotoxin-6b in aqueous solution determined by NMR spectroscopy and distance geometry.

The solution structure of sarafotoxin-6b in water has been determined using high-resolution NMR spectroscopy. 127 proton-proton distance measurements and three phi dihedral angle constraints derived from NMR spectra were used to calculate the solution structure using a combination of distance geometry and restrained molecular dynamics. The major structural feature of the resulting family of five structures was a right-handed alpha-helix extending from K9 to Q17. In contrast, the C-terminal region of the peptide appears not to adopt a preferred conformation in aqueous solution. The present structure is compared with those previously determined for endothelin peptides in non-aqueous solvents.

Shark myelin basic protein: amino acid sequence, secondary structure, and self-association.

Myelin basic protein (MBP) from the Whaler shark (Carcharhinus obscurus) has been purified from acid extracts of a chloroform/methanol pellet from whole brains. The amino acid sequence of the majority of the protein has been determined and compared with the sequences of other MBPs. The shark protein has only 44% homology with the bovine protein, but, in common with other MBPs, it has basic residues distributed throughout the sequence and no extensive segments that are predicted to have an ordered secondary structure in solution. Shark MBP lacks the triproline sequence previously postulated to form a hairpin bend in the molecule. The region containing the putative consensus sequence for encephalitogenicity in the guinea pig contains several substitutions, thus accounting for the lack of activity of the shark protein. Studies of the secondary structure and self-association have shown that shark MBP possesses solution properties similar to those of the bovine protein, despite the extensive differences in primary structure.

Solid-state 13C-NMR studies of the effects of sodium ions on the gramicidin A ion channel.

End-to-end helical dimers of gramicidin A form transmembrane pores in lipid bilayers, through which monovalent ions may pass. The groups within the peptide that interact with these ions have been studied by application of solid-state spectroscopic methods to a series of gramicidin A analogues synthesized with 13C in selected peptide carbonyl groups. The resonances of D-Leu10, D-Leu12 and D-Leu14 analogues were perturbed in the presence of 0.16 M sodium ions, whereas the resonances of the carbonyls of Gly2, Ala3, D-Leu4 and Val7, which are closer to the formylated N-terminal end of the peptide, were unaffected. The observed changes in chemical shift anisotropy are indicative of a change in orientation of the abovementioned leucine carbonyls.

Effect of acyl chain length on the structure and motion of gramicidin A in lipid bilayers.

The transmembrane ion transport properties of gramicidin A have previously been shown to dependent on the nature of its lipid environment. Solid-state NMR spectroscopic studies of 13C-labelled analogues of gramicidin in oriented multilayers of phosphatidylcholine have shown that variation of the lipid hydrocarbon chain length has no effect on the structure or orientation of the peptide backbone.

Determination of the structure of a membrane-incorporated ion channel. Solid-state nuclear magnetic resonance studies of gramicidin A.

Solid-state nuclear magnetic resonance (NMR) measurements on 13C-labeled analogues of the ion channel-forming peptide, gramicidin A, have been used to directly determine the structure of this peptide in lipid membranes. Seven gramicidin analogues, each labeled in a single carbonyl group of gly2, L-ala3, D-leu4, L-val7, D-leu10, D-leu12, or D-leu14 were synthesized by the solid-phase method. These gramicidin analogues were incorporated into aligned multilayers of dimyristoylphosphatidylcholine, or diether lipid bearing 14- or 16-carbon chains, at a 1:15 peptide:lipid mole ratio. Proton-enhanced, 13C, solid-state spectra were obtained at several temperatures and over a range of sample orientations with respect to the spectrometer magnetic field to permit accurate measurement of the chemical shift anisotropies. The observed anisotropies indicate that all of the labeled carbonyl bonds are oriented almost parallel to the molecular long axis and perpendicular to the lipid bilayer plane. These orientations are consistent with gramicidin forming a beta 6.3 single-strand helix that is oriented parallel to the methylene chains of the lipid molecules. Comparison of the linewidths from labeled residues that are in the innermost turn of the helix (gly2, ala3, and D-leu4), in the center of the molecule (val7), and in the turn nearest the lipid bilayer surface (D-leu10, D-leu12, and D-leu14) suggests that although the peptide behaves largely as a rigid barrel, segments of the peptide close to the membrane surface possess greater motional freedom. At temperatures above the gel-to-liquid crystalline transition temperature (Tc) the gramicidin molecules rotate, with a less than millisecond correlation time, about the bilayer normal: several degrees below Tc they become immobile on the NMR timescale, without change in the channel conformation. In the L beta' phase the linewidths of the D-leu10, D-leu'2, and D-leu" resonances become equal to those of the other labeled sites, indicating reduced but equivalent motion for all of the peptide carbonyl groups.

Osteosarcoma in a patient with Hutchinson-Gilford progeria.

A 13-year-old female with Hutchinson-Gilford progeria, who developed an osteosarcoma of the right chest wall, is reported. This is the first reported association of a malignant neoplasm with this syndrome.

A ten-year study of carcinoma of the pancreas.

During the 10 years from 1965 through 1974, 86 patients with adenocarcinoma of the pancreas were diagnosed or referred for treatment at the University of Oregon Health Sciences Center. The average age and survival in these patients is not significantly different from other published series. Only four patients had resectable tumors, and of these two survived over twelve months. Eight other patients also survived over twelve months, and no two of these had the same treatment regimens (although nine of the ten long-term survivors received some form of chemotherapy). Fourteen patients received a combination of chemotherapy and radiation therapy. While their average survival was only two months longer than that of the entire series, three were long-term survivors. Over 75% had significant palliation; this suggests that this treatment should be considered in selected cases.