Copper-mediated amyloid-beta toxicity is associated with an intermolecular histidine bridge

Amyloid-beta peptide (A beta) is pivotal to the pathogenesis of Alzheimer disease. Here we report the formation of a toxic A beta-Cu2+ complex formed via a histidine-bridged dimer, as observed at Cu2+/ peptide ratios of > 0.6:1 by EPR spectroscopy. The toxicity of the A beta-Cu2+ complex to cultured primary cortical neurons was attenuated when either the pi- or tau-nitrogen of the imidazole side chains of His were methylated, thereby inhibiting formation of the His bridge. Toxicity did not correlate with the ability to form amyloid or perturb the acyl-chain region of a lipid membrane as measured by diphenyl- 1,3,5-hexatriene anisotropy, but did correlate with lipid peroxidation and dityrosine formation. P-31 magic angle spinning solid-state NMR showed that A beta and A beta-Cu2+ complexes interacted at the surface of a lipid membrane. These findings indicate that the generation of the A beta toxic species is modulated by the Cu2+ concentration and the ability to form an intermolecular His bridge.

Sulfated galactans from Australian specimens of the red alga Phacelocarpus peperocarpos (Gigartinales, Rhodophyta)

Polysaccharides from the red alga Phacelocarpos peperocarpos were extracted with hot water, clarified, and precipitated with 2-propanol. The native preparation was highly sulfated (36.2% w/w). Alkali modification decreased the sulfate content by 2.0% w/w. The alkali-modified polysaccharide is composed mostly of galactose (Gal, 51 mol%) and 3,6-anhydrogalactose (AnGal, 41 mol%), with minor amounts of a mono-O-methylgalactose (MeGal, 1 mol%), xylose (Xyl, 6 mol%), and glucose (Glc, 1 mol%). The FTIR spectrum of the alkali-modified polysaccharide resembled K-carrageenan with absorption at 930 cm(-1) (indicative of AnGal) and 850 cm(-1) (Gal ii-sulfate). However, an additional, major band of absorption occurred at 820 cm(-1) indicating the presence of equatorial sulfate ester substitution at O-6 of Gal residues, A combination of linkage and C-13 NMR spectroscopic analyses showed that the polysaccharide was composed predominantly of a novel repeating-unit, O-beta-D-galactopyranosyl 4,6-disulfate)-(1 --> 4)-3,6-anhydro-alpha-D-galactopyranose. Minor structural variations also occurred, including alternative patterns of sulfation and the presence of terminal Xylp, The location of the terminal Xylp residues was not certain but evidence supported their attachment at O-3 of some 4-linked Galp residues. The cell-wall galactans remain unchanged during the life cycle of the alga. (C) 1996 Elsevier Science Ltd.

Anti-Group A Streptococcal Vaccine Epitope STRUCTURE, STABILITY, AND ITS ABILITY TO INTERACT WITH HLA CLASS II MOLECULES

Streptococcus pyogenes infections remain a health problem in several countries due to poststreptococcal sequelae. We developed a vaccine epitope (StreptInCor) composed of 55 amino acids residues of the C-terminal portion of the M protein that encompasses both T and B cell protective epitopes. The nuclear magnetic resonance (NMR) structure of the StreptInCor peptide showed that the structure was composed of two microdomains linked by an 18-residue alpha-helix. A chemical stability study of the StreptInCor folding/unfolding process using far-UV circular dichroism showed that the structure was chemically stable with respect to pH and the concentration of urea. The T cell epitope is located in the first microdomain and encompasses 11 out of the 18 alpha-helix residues, whereas the B cell epitope is in the second microdomain and showed no alpha-helical structure. The prediction of StreptInCor epitope binding to different HLA class II molecules was evaluated based on an analysis of the 55 residues and the theoretical possibilities for the processed peptides to fit into the P1, P4, P6, and P9 pockets in the groove of several HLA class II molecules. We observed 7 potential sites along the amino acid sequence of StreptInCor that were capable of recognizing HLA class II molecules (DRB1*, DRB3*, DRB4*, and DRB5*). StreptInCoroverlapping peptides induced cellular and humoral immune responses of individuals bearing different HLA class II molecules and could be considered as a universal vaccine epitope.

An investigation into the synthesis of polycarbazole squaraine derivatives

The polycondensation of squaric acid with 1,2-(9-Ethylcarbazol-3-yl)ethene and N-ethyliminostilbene in polyphosphoric acid yielded insoluble polymers which included substituted phosphate groups on the phenyl rings. The presence of phosphorus in these polymers was identified using solid-state P-31 NMR and EDAX techniques. Furthermore the phosphate groups were not ionic, hence no charge-balancing anions were present; Both polymers did not electrically conduct but exhibited dielectric breakdown values of 0.1 and 0.06 MV cm(-1) respectively.

Solution structure of the sodium channel antagonist conotoxin GS: A new molecular caliper for probing sodium channel geometry

Background: The venoms of Conus snails contain small, disulfide-rich inhibitors of voltage-dependent sodium channels. Conotoxin GS is a 34-residue polypeptide isolated from Conus geographus that interacts with the extracellular entrance of skeletal muscle sodium channels to prevent sodium ion conduction. Although conotoxin GS binds competitively with mu conotoxin GIIIA to the sodium channel surface, the two toxin types have little sequence identity with one another, and conotoxin GS has a four-loop structural framework rather than the characteristic three-loop mu-conotoxin framework. The structural study of conotoxin GS will form the basis for establishing a structure-activity relationship and understanding its interaction with the pore region of sodium channels. Results: The three-dimensional structure of conotoxin GS was determined using two-dimensional NMR spectroscopy. The protein exhibits a compact fold incorporating a beta hairpin and several turns. An unusual feature of conotoxin GS is the exceptionally high proportion (100%) of cis-imide bond geometry for the three proline or hydroxyproline residues. The structure of conotoxin GS bears little resemblance to the three-loop mu conotoxins, consistent with the low sequence identity between the two toxin types and their different structural framework. However, the tertiary structure and cystine-knot motif formed by the three disulfide bonds is similar to that present in several other polypeptide ion channel inhibitors. Conclusions: This is the first three-dimensional structure of a 'four-loop' sodium channel inhibitor, and it represents a valuable new structural probe for the pore region of voltage-dependent sodium channels. The distribution of amino acid sidechains in the structure creates several polar and charged patches, and comparison with the mu conotoxins provides a basis for determining the binding surface of the conotoxin GS polypeptide.

Cell-wall polysaccharides from Australian red algae of the family Solieriaceae (Gigartinales, Rhodophyta): Novel, highly pyruvated carrageenans from the genus Callophycus

Cell-wall polysaccharides from six species of red algae of the genus Callophycus were mainly galactans comprised predominantly of galactose (Gal) and 3,6-anhydrogalactose (AnGal), and were rich in pyruvate and sulfate. The Fourier Transform Infrared (FTIR) spectra of the polysaccharides superficially resembled that of alpha-carrageenan (composed of the repeating disaccharide carrabiose 2-sulfate), with major bands of absorption indicative of if-linked AnGal, axial 2-sulfate on 4-linked AnGal, and unsulfated, 3-linked Gal. The FTIR spectra of solutions of Callophycus polysaccharides in D2O-phosphate buffer displayed absorption, corresponding to the carboxylate anion of the pyruvate acetal substituent. Methylation analysis showed that 3,4,6-linked Galp (interpreted as 4,6-pyruvated, 3-linked Galp) and 2,4-linked AnGalp (interpreted as 4-linked AnGalp 2-sulfate) were the dominant links, together with significant quantities of 3-linked Galp. Proton-decoupled C-13 nuclear magnetic resonance (NMR) spectroscopy showed the polysaccharides to be composed predominantly of pyruvated carrageenans. The C-13 NMR spectra were completely assigned by a J-modulated spin-echo pulse sequence and 2D experiments employing gradient Heteronuclear Multiple Bond Correlation (HMBC), C-13/H-1 Heteronuclear Multiple Quantum Coherence (HMQC), and HMQC Total Correlation Spectroscopy (HMQC-TOCSY). The Callophycus galactans thus consist predominantly of the novel repeating disaccharide 4',6'-O-(1-carboxyethylidene)carrabiose 2-sulfate and minor amounts of the alpha-carrageenan repeating unit (carrabiose 2-sulfate), and other structural variations. (C) 1997 Elsevier Science Ltd.

Metabolic activation of aromatic amines by human pancreas

Epidemiologic studies have suggested that aromatic amines (and nitroaromatic hydrocarbons) may be carcinogenic for human pancreas, Pancreatic tissues from 29 organ donors (13 smokers, 16 non-smokers) were examined for their ability to metabolize aromatic amines and other carcinogens, Microsomes showed no activity for cytochrome P450 (P450) 1A2-dependent N-oxidation of 4-aminobiphenyl (ABP) or for the following activities (and associated P450s): aminopyrine N-demethylation and ethylmorphine N-demethylation (P450 3A4); ethoxyresorufin O-deethylation (P450 1A1) and pentoxyresorufin O-dealkylation (P450 2B6); p-nitrophenol hydroxylation and N-nitrosodimethylamine N-demethylation (P450 2E1); lauric acid omega-hydroxylation (P450 4A1); and 4-(methylnitrosamino)-1-(3-pyridyl-1-butanol) (NNAL) and 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK) alpha-oxidation (P450 1A2, 2A6, 2D6). Antibodies were used to examine microsomal levels of P450 1A2, 2A6, 2C8/9/18/19, 2E1, 2D6, and 3A3/ 4/5/7 and epoxide hydrolase. Immunoblots detected only epoxide hydrolase at low levels; P450 levels were <1% of liver. Microsomal benzidine/prostaglandin hydroperoxidation activity was low. In pancreatic cytosols and microsomes, 4-nitrobiphenyl reductase activities were present at levels comparable to human liver. The O-acetyltransferase activity (AcCoA-dependent DNA-binding of [H-3]N-hydroxy-ABP) of pancreatic cytosols was high, about two-thirds the levels measured in human colon. Cytosols showed high activity for N-acetylation of p-aminobenzoic acid, but not of sulfamethazine, indicating that acetyltransferase-1 (NAT1) is predominantly expressed in this tissue. Cytosolic sulfotransferase was detected at low levels. Using P-32-post-labeling enhanced by butanol extraction, putative arylamine-DNA adducts were detected in most samples. Moreover, in eight of 29 DNA samples, a major adduct was observed that was chromatographically identical to the predominant ABP-DNA adduct, N-(deoxyguanosin-8-yl)-ABP. These results are consistent with a hypothesis that aromatic amines and nitroaromatic hydrocarbons may be involved in the etiology of human pancreatic cancer.

The role of xenobiotic metabolizing enzymes in arylamine toxicity and carcinogenesis: Functional and localization studies

In both animal models and humans, the first and obligatory step in the activation of arylamines is N-hydroxylation. This pathway is primarily mediated by the phase-I enzymes CYP1A1, CYP1A2 and CYP4B1. In the presence of flavonoids such as alpha-naphthoflavone and flavone, both CYP3A4 and CYP3A5 have also been shown to play a minor role in the activation of food-derived heterocyclic amines. The further activation of N-hydroxyarylamines by phase-II metabolism can involve both N,O-acetylation and N,O-sulfonation catalyzed by N-acetyltransferases (NAT1 and NAT2) and sulfotransferases, respectively. Using an array of techniques, we have been unable to detect constitutive CYP1A expression in any segments of the human gastrointestinal tract. This is in contrast to the rabbit where CYP1A1 protein was readily detectable on immunoblots in microsomes prepared from the small intestine. In humans, CYP3A3/3A4 expression was detectable in the esophagus and all segments of the small intestine. Northern blot analysis of eleven human colons showed considerable heterogeneity in CYP3A mRNA between individuals, with the presence of two mRNA species in same subjects. Employing the technique of hybridization histochemistry (also known as in situ hybridization), CYP4B1 expression was observed in some human colons but not in the liver or the small intestine. Hybridization histochemistry studies have also demonstrated variable NAT1 and NAT2 expression in the human gastrointestinal tract. NAT1 and NAT2 mRNA expression was detected in the human liver, small intestine, colon, esophagus, bladder, ureter, stomach and lung. Using a general aryl sulfotransferase riboprobe (HAST1), we have demonstrated marked sulfotransferase expression in the human colon, small intestine, lung, stomach and liver. These studies demonstrate that considerable variability exists in the expression of enzymes involved in the activation of aromatic amines in human tissues. The significance of these results in relation to a role for heterocyclic amines in colon cancer is discussed.

Molecular motion in miscible polymer blends .1. Motion in blends of PEO and PVPh studied by solid-state C-13 T-1 rho measurements

Changes in molecular motion in blends of PEO-PVPh have been studied using measurements of C-13 T-1 rho relaxation times. C-13 T-1 rho relaxation has been confirmed as arising from spin-lattice interactions by observation of the variation in T-1 rho with rf field strength and temperature. In the pure homopolymers a minimum in T-1 rho is observed at ca. 50 K above the glass transition temperatures detected by DSC. After blending, the temperature of the minimum in T-1 rho for PEO increased, while that for PVPh decreased, however, the minima, which correspond to the temperatures where the average correlation times for reorientation are close to 3.1 mu s, are separated by 45 K (in a 45% PEO-PVPh blend). These phenomena are explained in terms of the local nature of T-1 rho measurements. The motions of the individual homopolymer chains are only partially coupled in the blend. A short T-1 rho has been observed for protonated aromatic carbons, and assigned to phenyl rings undergoing large-angle oscillatory motion, The effects of blending, and temperature, on the proportion of rings undergoing oscillatory motion are analyzed.

The structure of a novel insecticidal neurotoxin, omega-atracotoxin-HV1, from the venom of an Australian funnel web spider

A family of potent insecticidal toxins has recently been isolated from the venom of Australian funnel web spiders. Among these is the 37-residue peptide omega-atracotoxin-HV1 (omega-ACTX-HV1) from Hadronyche versuta. We have chemically synthesized and folded omega-ACTX-HV1, shown that it is neurotoxic, ascertained its disulphide bonding pattern, and determined its three-dimensional solution structure using NMR spectroscopy. The structure consists of a solvent-accessible beta-hairpin protruding from a disulphide-bonded globular core comprising four beta-turns. The three intramolecular disulphide bonds form a cystine knot motif similar to that seen in several other neurotoxic peptides. Despite limited sequence identity, omega-ACTX-HV1 displays significant structural homology with the omega-agatoxins and omega-conotoxins, both of which are vertebrate calcium channel antagonists; however, in contrast with these toxins, we show that omega-ACTX-HV1 inhibits insect, but not mammalian, voltage-gated calcium channel currents.

Conformational dynamics of thyroid hormones by variable temperature nuclear magnetic resonance: The role of side chain rotations and cisoid/transoid interconversions

H-1 NMR spectra of the thyroid hormone thyroxine recorded at low temperature and high field show splitting into two peaks of the resonance due to the H2,6 protons of the inner (tyrosyl) ring. A single resonance is observed in 600 MHz spectra at temperatures above 185 K. An analysis of the line shape as a function of temperature shows that the coalescence phenomenon is due to an exchange process with a barrier of 37 kJ mol(-1). This is identical to the barrier for coalescence of the H2',6' protons of the outer (phenolic) ring reported previously for the thyroid hormones and their analogues. It is proposed that the separate peaks at low temperature are due to resonances for H2,6 in cisoid and transoid conformers which are populated in approximately equal populations. These two peaks are averaged resonances for the individual H2 and H6 protons. Conversion of cisoid to transoid forms can occur via rotation of either the alanyl side chain or the outer ring, from one face of the inner ring to the other. It is proposed that the latter process is the one responsible for the observed coalescence phenomenon. The barrier to rotation of the alanyl side chain is greater than or equal to 37 kJ mol(-1), which is significantly larger than has previously been reported for Csp(2)-Csp(3) bonds in other Ph-CH2-X systems. The recent crystal structure of a hormone agonist bound to the ligand-binding domain of the rat thyroid hormone receptor (Wagner et al. Nature 1995, 378, 690-697) shows the transoid form to be the bound conformation. The significant energy barrier to cisoid/transoid interconversion determined in the current study combined with the tight fit of the hormone to its receptor suggests that interconversion between the forms cannot occur at the receptor site but that selection for the preferred bound form occurs from the 50% population of the transoid form in solution.

Direct visualisation of B-1 inhomogeneity by flip angle dependency

Inhomogeneities in the spatial distribution of the excitatory Radio Frequency (RF) field, are still a dominant source of artifacts and loss of signal to noise ratio in MR imaging experiments, A number of strategies have been proposed to quantify this distribution, However, in this technical note we present a relatively simple MR imaging procedure which can be used to visualise RF inhomogeneities directly either by means of the magnitude or the phase of an image. To visualise the RF field distribution in both the inner and outer volumes of the coil, we have performed experiments in which the entire coil is submerged in a non-conducting fluid, To the best of our knowledge this strategy has not been used previously in order to evaluate coil performance, Finally, we demonstrate that the method is sensitive enough to reveal the effects of the sample properties on the effective RF wavelength of the transmitted field. (C) 1997 Elsevier Science Inc.

Structure of galactosylononitol

A cyclitol galactoside was isolated from the seeds of Vigna angularis and shown to be identical with galactosylononitol. However, detailed 2D NMR data and methylation analysis resulted in the revision of the structure of galactosylononitol to O-alpha-D-galactopyranosyl-(1-->3)-4-O-methyl-D-myo-inositol (1). Thus, compound 1 represents the only naturally occuring methylated derivative of galactinol.