Alcohol Selectivity of β3-Containing GABAA Receptors: Evidence for a Unique Extracellular Alcohol/Imidazobenzodiazepine Ro15-4513 Binding Site at the α+β- Subunit Interface in αβ3δ GABAA Receptors

GABA_A receptors (GABARs) have long been the focus for acute alcohol actions with evidence for behaviorally relevant low millimolar alcohol actions on tonic GABA currents and extrasynaptic α4/6, δ, and β3 subunit-containing GABARs. Using recombinant expression in oocytes combined with two electrode voltage clamp, we show with chimeric β2/β3 subunits that differences in alcohol sensitivity among β subunits are determined by the extracellular N-terminal part of the protein. Furthermore, by using point mutations, we show that the β3 alcohol selectivity is determined by a single amino acid residue in the N-terminus that differs between GABAR β subunits (β3Y66, β2A66, β1S66). The β3Y66 residue is located in a region called “loop D” which in γ subunits contributes to the imidazobenzodiazepine (iBZ) binding site at the classical α+γ2- subunit interface. In structural homology models β3Y66 is the equivalent of γ2T81 which is one of three critical residues lining the benzodiazepine binding site in the γ2 subunit loop D, opposite to the “100H/R-site” benzodiazepine binding residue in GABAR α subunits. We have shown that the α6R100Q mutation at this site leads to increased alcohol-induced motor in-coordination in alcohol non-tolerant rats carrying the α6R100Q mutated allele. Based on the identification of these two amino acid residues α6R100 and β66 we propose a model in which β3 and δ containing GABA receptors contain a unique ethanol site at the α4/6+β3- subunit interface. This site is homologous to the classical benzodiazepine binding site and we propose that it not only binds ethanol at relevant concentrations (EC₅₀–17 mM), but also has high affinity for a few selected benzodiazepine site ligands including alcohol antagonistic iBZs (Ro15-4513, RY023, RY024, RY80) which have in common a large moiety at the C7 position of the benzodiazepine ring. We suggest that large moieties at the C7-BZ ring compete with alcohol for its binding pocket at a α4/6+β3- EtOH/Ro15-4513 site. This model reconciles many years of alcohol research on GABARs and provides a plausible explanation for the competitive relationship between ethanol and iBZ alcohol antagonists in which bulky moieties at the C7 position compete with ethanol for its binding site. We conclude with a critical discussion to suggest that much of the controversy surrounding this issue might be due to fundamental species differences in alcohol and alcohol antagonist responses in rats and mice.     

A new naphthalene derivative with anti-amyloidogenic activity as potential therapeutic agent for Alzheimer's disease

The aggregation of β-amyloid peptides is associated to neurodegeneration in Alzheimer’s disease (AD) patients. Consequently, the inhibition of both oligomerization and fibrillation of β-amyloid peptides is considered a plausible therapeutic approach for AD. Herein, the synthesis of new naphthalene derivatives and their evaluation as anti-β-amyloidogenic agents are presented. Molecular dynamic simulations predicted the formation of thermodynamically stable complexes between the compounds, the Aβ_1-42 peptide and fibrils. In human microglia cells, these compounds inhibited the aggregation of Aβ_1-42 peptide. The lead compound 8 showed a high affinity to amyloid plaques in mice brain ex vivo assays and an adequate log P_oct/PBS value. Compound 8 also improved the cognitive function and decreased hippocampal β-amyloid burden in the brain of 3xTg-AD female mice. Altogether, our results suggest that 8 could be a novel therapeutic agent for AD.     

Bimolecular fluorescence complementation and targeted biotinylation provide insight into the topology of the skeletal muscle Ca2+ channel β1a subunit

In skeletal muscle, L-type calcium channels (DHPRs), localized to plasma membrane sarcoplasmic reticulum junctions, are tightly packed into groups of four termed tetrads. Here, we have used bimolecular fluorescence complementation (BiFC) and targeted biotinylation to probe the structure and organization of β1a subunits associated with native Ca_V1.1 in DHPRs of myotubes. The construct YN-β1a-YC, in which the non-fluorescent fragments of YFP (“YN” corresponding to YFP residues 1–158, and “YC” corresponding to YFP residues 159–238) were fused, respectively, to the N- and C-termini of β1a, was fully functional and displayed yellow fluorescence within DHPR tetrads after expression in β1-knockout (β1KO) myotubes; this yellow fluorescence demonstrated the occurrence of BiFC of YN and YC on the β1a N- and C-termini. In these experiments, we avoided overexpression because control experiments in non-muscle cells indicated that this could result in non-specific BiFC. BiFC of YN-β1a-YC in DHPR tetrads appeared to be intramolecular between N- and C-termini of individual β1a subunits rather than between adjacent DHPRs because BiFC (1) was observed for YN-β1a-YC co-expressed with Ca_V1.2 (which does not form tetrads) and (2) was not observed after co-expression of YN-β1a-YN plus YC-β1a-YC in β1KO myotubes. Thus, β1a function is compatible with N- and C-termini being close enough together to allow BiFC. However, both termini appeared to have positional freedom and not to be closely opposed by other junctional proteins since both were accessible to gold-streptavidin conjugates. Based on these results, a model is proposed for the arrangement of β1a subunits in DHPR tetrads.     

C-Terminal Turn Stability Determines Assembly Differences between Aβ40 and Aβ42

Oligomerization of the amyloid β-protein (Aβ) is a seminal event in Alzheimer's disease. Aβ42, which is only two amino acids longer than Aβ40, is particularly pathogenic. Why this is so has not been elucidated fully. We report here results of computational and experimental studies revealing a C-terminal turn at Val36–Gly37 in Aβ42 that is not present in Aβ40. The dihedral angles of residues 36 and 37 in an Ile31–Ala42 peptide were consistent with β-turns, and a β-hairpin-like structure was indeed observed that was stabilized by hydrogen bonds and by hydrophobic interactions between residues 31–35 and residues 38–42. In contrast, Aβ(31–40) mainly existed as a statistical coil. To study the system experimentally, we chemically synthesized Aβ peptides containing amino acid substitutions designed to stabilize or destabilize the hairpin. The triple substitution Gly33Val–Val36Pro–Gly38Val (“VPV”) facilitated Aβ42 hexamer and nonamer formation, while inhibiting formation of classical amyloid-type fibrils. These assemblies were as toxic as were assemblies from wild-type Aβ42. When substituted into Aβ40, the VPV substitution caused the peptide to oligomerize similarly to Aβ42. The modified Aβ40 was significantly more toxic than Aβ40. The double substitution d-Pro36–l-Pro37 abolished hexamer and dodecamer formation by Aβ42 and produced an oligomer size distribution similar to that of Aβ40. Our data suggest that the Val36–Gly37 turn could be the sine qua non of Aβ42. If true, this structure would be an exceptionally important therapeutic target.     

Studies on the structure and mechanism of action of glycoside hydrolases. I. Purification and study of some factors affecting the activity of Rhizopus arrhizus (1 leads to 3)-beta-D-glucanase

The extracellular (1→3)-β-D-glucanase [(1→3)-β-D-glucan glucanohydrolase, E.C. 3.2.1.6] produced by Rhizopus arrhizzus QM 1032 was purified 165-fold by chromatography. The purified enzyme is basic, has a molecular weight of ≈ 10,000, and is unstable in dilute solution but may be stabilized by addition of human serum albumin. The pH-activity profile for the enzyme in the presence of serum albumin shows a peak at about pH 3.5–3.7 and a shoulder at pH 4.5–4.6, whereas in the absence of serum albumin the optimum pH is at pH 4.5–4.6, indicating the presence of two enzymic species, designated “pH 3. 5 activity” and “pH 4.6 activity”. In the presence of albumin the enzyme activity is resistant to inactivation by a wide range of reagents. Ammonium molybdate is, however, a powerful inhibitor of ldpH 3.5 activity” although a much poorer inhibitor of “pH 4.6 activity”. The enzyme activity is stable during heating at pH 3.5 in the presence of human serum albumin. Thus, 94.5 and 88.5 % of “pH 3.5 activity” and “pH 4.6 activity”, respectively, remained after heat treatment for 30 min at 68°. The enzyme is, however, essentially inactive at this temperature, even in the presence of albumin. To account for this finding, a temperature-dependent conformational change is proposed. The enzyme activity is not stable during heating at pH 4.6 in the presence of serum albumin. K_m values for action on laminaran are 0.54 mg/ml (pH 3.5) and 0.27 mg/ml (pH 4.6). For lichenan the corresponding values are 3.33 and 2.38 mg/ml. The V_max for enzyme action on lichenan is 35–40% higher than for action on laminaran at both pH values. Possible relationships between the two forms of the enzyme are briefly considered.     

De novo design,synthesis and solution conformational study of two didehydroundecapeptides: effect of nature and number of amino acids interspersed between Phe residues

De novo design of peptides and proteins has recently surfaced as an approach for investigating protein structure and function. This approach vitally tests our knowledge of protein folding and function, while also laying the groundwork for the fabrication of proteins with properties not precedented in nature. The success relies heavily on the ability to design relatively short peptides that can espouse stable secondary structures. To this end, substitution with α,β‐didehydroamino acids, especially α,β‐didehydrophenylalanine (Δ^zPhe), comes in use for spawning well‐defined structural motifs. Introduction of ΔPhe induces β‐bends in small and 3₁₀‐helices in longer peptide sequences. The present work aims to investigate the effect of nature and the number of amino acids interspersed between two ΔPhe residues in two model undecapeptides, Ac‐Gly‐Ala‐ΔPhe‐Ile‐Val‐ΔPhe‐Ile‐Val‐ΔPhe‐Ala‐Gly‐NH₂ (I) and Boc‐Val‐ΔPhe‐Phe‐Ala‐Phe‐ΔPhe‐Phe‐Leu‐Ala‐ΔPhe‐Gly‐OMe (II). Peptide I was synthesized using solid‐phase chemistry and characterized using circular dichroism spectroscopy. Peptide II was synthesized using solution‐phase chemistry and characterized using circular dichroism and nuclear magnetic resonance spectroscopy. Peptide I was designed to examine the effect of incorporating β‐strand‐favoring residues like valine and isoleucine as spacers between two ΔPhe residues on the final conformation of the resulting peptide. Circular dichroism studies on this peptide have shown the existence of a 3₁₀‐helical conformation. Peptide II possesses three amino acids as spacers between ΔPhe residues and has been reported to adopt a mixed 3₁₀/α‐helical conformation using circular dichroism and nuclear magnetic resonance spectroscopy studies. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Multivalent Ligand System Carrying Enkephalin and Neurotensin Coimmobilized on Liposomes

A multivalent ligand system was constructed by coimmobilization of two kinds of peptide ligands, enkephalin and neurotensin derivatives having a dioctadecyl group, on dimyristoylphosphatidylcholine (DMPC) liposomes. The enkephalin derivatives are Tyr-D -Ala-Gly-Trp-Leu- (Sar-Sar-Pro)_n-[N(C₁₈H₃₇)₂] (Enk3nD, n=0, 1, 2), where a dioctadecyl group was connected to the C-terminal side of enkephalin directly or through a hydrophilic and flexible spacer chain of different lengths. The neurotensin derivatives are Ac-Glu[N(C₁₈H₃₇)₂]-(Sar-Sar-Pro)_n-Arg-Arg-Pro-Tyr-Ile-Leu-OH (D3nNT, n=0, 1, 2, 3). The derivatives were spontaneously immobilized on DMPC liposomes by overnight incubation. The receptor affinity of the enkephalin derivatives became significantly higher upon immobilization on liposomes. The highest affinity was obtained for the δ receptor by Enk6D immobilized on DMPC liposomes. This affinity is higher than that of enkephalinamide. Neurotensin derivatives coimmobilized with large amounts of Enk3D on DMPC liposomes show higher affinity than the neurotensin derivatives immobilized alone. The effect of Enk3D on the receptor affinity of the coimmobilized neurotensin derivative disappeared by the addition of [Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAGO). Therefore, the receptor affinity of a peptide hormone is altered by immobilization on DMPC liposomes and by coimmobilization with other peptide hormones. It was confirmed by fluorescent microscopy that the multivalent ligand system binds to receptors without release of the bound ligands from DMPC liposomes.     

Synthesis and stability assay of 4-methylumbelliferyl (1→3)-β-D-pentaglucoside

The synthesis and stability of 4-methylumbelliferyl (1 → 3)-β-D-pentaglucoside 3 are described. The (1 → 3)-β-D-glucan isolated from the cell walls of Saccharomyces cerevisiae was recovered from the aqueous medium as water-insoluble particles by the spray drying (GS) method. The acid-solubilized (1 → 3)-β-D-oligoglucosides were prepared by partial acid hydrolysis of glucan. The peracetylated (1 → 3)-β-D-pentaglucoside 1 was obtained by isolation of peracetylated (1 → 3)-β-D-oligoglucoside mixture. The peracetylated 4-methylumbelliferyl (1 → 3)-β-D-pentaglucoside 2 was synthesized by treating compound 1 with the 4-methylumbelliferone and a Lewis acid (SnCl₄) catalyst. NaOMe in dry methanol was used for the deacetylation of the blocked derivative, to give the target compound 3 in an overall yield of 35%. Activity assays with β-glucosidase indicated that compound 3 was much more stable than the corresponding pentasaccharide.     

Contrasting solution conformations of peptides containing α,α-dialkylated residues with linear and cyclic side chains

The conformational properties of α,α-dialkylated amino acid residues possessing acyclic (diethylglycine, Deg: di-n-propylglycine, Dpg; di-n-butylglycine, Dbg) and cyclic (1-amino-cycloalkane-1-carboxylic acid, Ac_nc) side chains have been compared in solution. The five peptides studied by nmr and CD spectroscopy are Boc-Ala-Xxx-Ala-OMe, where Xxx = Deg(I). Dpg (II), Dbg (III), Ac₆c (IV), and Ac₇c (V). Delineation of solvent-shielded NH groups have been achieved by solvent and temperature dependence of NH chemical shifts in CDCl₃ and (CD₃)₂SO and by paramagnetic radical induced line broadening in pepiide III. In the Dxg peptides the order of solvent exposure of NH groups is Ala(1) > Ala(3) > Dxg(2), whereas in the Ac_nc peptides the order of solvent exposure of NH groups is Ala(1) > Ac_nc(2) > Ala(3). The nmr results suggest that Ac_nc peptides adopt folded β-turn conformations with Ala(1) and Ac_nc(2) occupying i + 1 and i + 2 positions. In contrast, the Dxg peptides favor extended C₅ conformations. The conformational differences in the two series are clearly borne out in CD studies. The solution conformations of peptides I-III are distinctly different from the β-turn structure observed in crystals. Low temperature nmr spectra recorded immediately after dissolution of crystals of peptide II provide evidence for a structural transition. Introduction of an additional hydrogen-bonding function in Boc-Ala-Dpg-Ala-NHMe (VI) results in a stabilization of a consecutive β-turn or incipient 3₁₀-helix in solution. © 1995 John Wiley & Sons, Inc.     

Local secondary structure in ribonuclease A denatured by guanidine · HCl near 1 °C

Recent work has shown that a short α-helix can be stable in water near 1 °C when stabilized by specific interactions between side-chains, while earlier “host-guest” results with random copolymers have shown that a short α-helix is unstable in water at all temperatures in the absence of stabilizing side-chain interactions. As regards the mechanism of protein folding, it is now reasonable on energetic grounds to consider isolated α-helices and β-sheets as the first intermediates on the pathway of protein folding. Proton nuclear magnetic resonance is used here to detect isolated secondary structures in ribonuclease A denatured by guanidine · HCl (GuHCl). Temperatures near 1 °C are used because the low-temperature stability of the C-peptide helix may be a general property of isolated secondary structures in water.Our procedure is to titrate with GuHCl the C2H resonance lines of the four histidine residues of denatured ribonuclease A. Studies of model peptides (C-peptide (lactone) and C-peptide carboxylate, residues 1 to 13 of ribonuclease A; S-peptide, residues 1 to 20) show linear titration curves for the C2H resonance of His12 above 0.5 M-GuHCl, once helix unfolding is complete. Deviations from this line are used to monitor helix formation. The GuHCl titration curves of the other three histidine residues are also linear, once unfolding is complete. The results show that the helix found in C-peptide and S-peptide is also found in denatured ribonuclease A, where it behaves as an isolated helix not stabilized significantly by interactions with other chain segments. Studies of denatured S-protein show that the remaining three His residues, His48, His105 and His119, are involved in structure only below 1 m-GuHCl at 9 °C, pH 1.9. The nature of this structure is not known. The main conclusion from this work is that the His12 helix can be observed as a stable, isolated helix in denatured ribonuclease A near 1 °C, and that none of the other three His residues is involved in a comparably stable local structure. In native ribonuclease A, His12 is within an α-helix and the other three His residues are involved in a 3-stranded β-sheet structure.The helix-coil transition of C-peptide has also been studied for other side-chain resonances by GuHCl titration. Typically, but not always, the titration curves are linear after helix unfolding takes place and resonance lines from different residues of the same amino acid type can be resolved in GuHCl solutions. This is true of the four histidine residues of ribonuclease A although their pK values in 5 m-GuHCl are nearly the same. In C-peptide, the βCH₃ resonance of Ala6 is affected strongly by GuHCl while the lines of Ala4 and Ala5 are shifted only weakly by GuHCl. Evidently the interactions between GuHCl and side-chains in an unfolded peptide depend upon neighboring groups.     

β-Alanine containing cyclic peptides with turned structure: The “pseudo type II β-turn.” VI

In the present paper we describe the synthesis, purification, single crystal x-ray analysis, and nmr solution characterization, combined with restrained molecular dynamic simulations, of the cyclic hexapeptide cyclo-(L -Pro-L -Phe-β-Ala)₂. The peptide was synthesized by classical solution methods and the cyclization of the free hexapeptide was accomplished in good yields in diluted methylene chloride solution using N,N-dicyclohexyl-carbodiimide. The compound crystallizes in the monoclinic space group P2₁ from methanol-dichloro-methane solution. The two identical halves of the molecule adopt in the solid state two different conformations. One β-Ala-L -Pro peptide bond is trans, while the second is cis. The molecule is present in dimethylsulfoxide d₆ solutions as a mixture of conformational families. One of these corresponds to a C₂ symmetrical molecule with both β-Ala-Pro cis peptide bonds, while the second major conformation is very similar to that observed in the solid state. All Pro-Phe segments, both in the solid state and the symmetrical and unsym-metrical solution conformations, display ?,ψ angles close to that of position i + 1 and i + 2 of type II β-turns. In addition, the segments preceeded by a trans β-Ala-Pro peptide bond are characterized by a typical i ← i + 3 hydrogen bond, which is absent in the conformer containing a cis β-Ala-Pro peptide bond. The latter conformation corresponds to a new structural domain we define as the “pseudo type II β-turn.” © 1994 John Wiley & Sons, Inc.     

Synthesis and conformational analysis of N-glycopeptides. II. CD,molecular dynamics,and nmr spectroscopic studies on linear N-glycopeptides

The comprehensive structural analysis reported herein of eight N-glycopeptides, in three different solvents, is based on quantitative CD experiments, homonuclear nuclear Overhauser effect measurements, and molecular dynamics (MD) calculations. Although several orientations of the two amide planes attached to the carbohydrate pyranose ring are possible, according to NOE, CD data, and MD simulations, of all of the glycopeptide models, regardless of the type of the carrier peptide, only one dominant conformer population was found. This conformer is characterized by a nearly trans orientation of the CH and NH hydrogens of both acetamido groups. This finding is in perfect agreement with x-ray crystallographic data on the solid state conformation of the 1-N-acetyl- and 1-N-(β-aspartoyl)-2-acetamido-2-deoxy-β-D -glucopyranosylamine. The precise identification of this dominant conformer of N-glycopeptides in solution was the major question addressed herein by the structural analyses. A “CD additivity” experiment was carried out using an equimolar solution of Boc-Pro-Asp-NHCH₃ and l-N-acetyl-3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-D -gluco-pyranosylamine at ambient temperature in acetonitrile. The CD spectrum obtained from the equimolar solution of the above two molecules (the “spectroscopic sum”) was identical with the CD curve obtained from the algebraic summation of the individually recorded CD spectra of the peptide and the carbohydrate moiety (“mathematical sum”). The global picture of the CD spectral analyses of the eight parent peptides with the eight N-glycopeptides revealed that in trifluoroethanol and acetonitrile, the side-chain modification of the Asn models (natural N-glycopeptide analogues) by N-glycosylation has a significant effect on the conformation of the carrier peptide, resulting in a decrease in the original type I β-turn content. Simultaneously, the type II β-turn conformational percentage increased to ≈ 20%. Such a conformational ratio change seems to be larger than the expected errors arising from the CD analyses, and agrees with the results of MD calculations. N-glycosylation of Asn residues causes perturbations, not only through the covalent bond, but also through specific hydrogen bonds between the backbone and side chain atoms. CD spectroscopy, augmented by efficient CD curve deconvolution techniques, has proved to be a useful tool for studying multicomponent conformer mixtures of small linear peptides in solution and changes of conformational equilibria caused by N-glycosylation. © 1993 John Wiley & Sons, Inc.     

Expression of β1,4-galactosyltransferase in the development of mouse brain

β_1,4-Galactosyltransferase (GalTase, EC 2.4.1.38) transfers galactose to the terminal N-acetylglucosamine of complex-type N-glycans, which have great importance for cell-cell interactions during fertilization and early embryogenesis. In this study, the activity of β_1,4-galactosyltransferase in mouse brain during development was measured with the method of reverse HPLC using a fluorescence-labeled biantenary sugar chain, GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3) Manβ1-4GlcNAcβ1-4GlcNAc-PA. The level of messenger RNA of this enzyme during the development of mouse brain was also investigated with Northern blot analysis. The results showed that: (1) β_1,4-galactosyltransferase showed similar branch specificity and kinetics for the biantenary substrate during development; (2) GalTase activity in fetal mouse brain was four times higher than that in adult mouse brain and decreased gradually in the course of development; (3) messenger RNA level was highest in fetal mouse and decreased dramatically after birth. However, the contents of mRNA were not parallel to the enzyme activity.     

Unique assignment of inter-subunit association in GABAA α1β3γ2 receptors determined by molecular modeling

Recent publications defined requirements for inter-subunit contacts in a benzodiazepine-sensitive GABA_A receptor (GABA_ARα1β3γ2). There is strong evidence that the heteropentameric receptor contains two α1, two β3, and one γ2 subunit. However, the available data do not distinguish two possibilities: When viewed clockwise from an extracellular viewpoint the subunits could be arranged in either γ2β3α1β3α1 or γ2α1β3α1β3 configurations. Here we use molecular modeling to thread the relevant GABA_AR subunit sequences onto a template of homopentameric subunits in the crystal structure of the acetylcholine binding protein (AChBP). The GABA_A sequences are known to have 15-18% identity with the acetylcholine binding protein and nearly all residues that are conserved within the nAChR family are present in AChBP. The correctly aligned GABA_A sequences were threaded onto the AChBP template in the γ2β3α1β3α1 or γ2α1β3α1β3  arrangements. Only the γ2α1β3α1β3 arrangement satisfied three known criteria: (1) α1 His¹⁰² binds at the γ2 subunit interface in proximity to γ2 residues Thr¹⁴², Phe⁷⁷, and Met¹³⁰; (2) α1 residues 80-100 bind near γ2 residues 91-104; and (3) α1 residues 58-67 bind near the β3 subunit interface. In addition to predicting the most likely inter-subunit arrangement, the model predicts which residues form the GABA and benzodiazepine binding sites.     

Release in vivo of Met-enkephalin and encrypted forms of Met-enkephalin from brain and spinal cord of the anesthetized cat

Processing of the proenkephalin molecule will result in peptide fragments in which the pentapeptide YGGFM is included. We have employed a molecular sieve (2 kDa) separation, enzyme hydrolysis radioimmunoassay (RIA) treatment sequence which permits concurrent measurement of Met-enkephalin (Enk) and several enkephalin-encrypting (X-Enk) peptides in a single sample. Using this protocol, the release of Enk and X-Enk (total Enk - Enk) greater and less than 2 kDa from spinal cord and the mesencephalic aqueductal grey was assessed under resting conditions and during stimulation of the sciatic nerve in the chloralose-urethane anesthetized cat. Under resting conditions measurable levels of Enk (10.5±4.7; 9.1±2.1 pg/min) and X-Enk (47.8±19.7; 45.7±12.3 pg/min) are found in aqueductal and spinal superfusates, respectively. The X-Enk measured under resting and evoked conditions in aqueductal and spinal perfusates is associated almost exclusively (>90–95%) with fragments >2 kDa in size. These results, showing the relative absence of detectable levels of X-Enk forms <2 kDa, were confirmed by reverse phase chromatography. During sciatic nerve stimulation, the levels of both Enk and X-Enk were mildly elevated in spinal and ventricular perfusates. With the addition of thiorphan (10⁻⁵ M), though there was no effect on the resting release of either Enk or X-Enk, the levels of Enk measured under evoked conditions were significantly augmented in both ventricular and spinal perfusates.     

Expressions of some antioxidant genes in SH-SY5Y cells treated with β-lapachone,morphine and electromagnetic field

β-Lapachone (β-Lap), morphine (Mor), and electromagnetic field (EMF) generate reactive oxygen species. The goal of the present study was to examine the effects of Mor and EMF, in combination with β-Lap on the cell growth inhibition and expression of several antioxidant genes. The 0.50 mT intensity of 50 Hz EMF and two exposure conditions (“15 min field-on/15 min field-off” and “30 min field-on continuously”) on SH-SY5Y cells were used. The effects of Mor and EMF, in combination with β-Lap on cell growth inhibition and the expression levels of several antioxidant genes (NQO1, NQO2, SOD1, SOD2, CAT, GSTO1, GSTM2, GSTM3, GSTP1, MGST1, MGST3) in SH-SY5Y cells were measured. The relative mRNA levels were calculated according to the \({{\text{2}}^ - }^{{\Delta \Delta {{\text{C}}_{\text{t}}}}}\). Whereas NQO1 mRNA level decreased in the “15 min field-on/15 min field-off” condition, the expression level of NQO2 was increased. Both NQO1 and NQO2 expressions increased in Mor treated cells. IC₅₀ values of β-Lap in combination with Mor, EMF, and “Mor?+?EMF” were higher than cells treated only with β-Lap. The NQO1 expression level in the cells treated with β-Lap was higher than the other treatments, indicating that β-Lap induces the expression of NQO1. Moreover, multiple linear regression analysis indicated that NQO1 mRNA levels were associated positively with β-Lap and negatively with EMF. At least in part, the mRNA levels of NQO1 were associated with IC₅₀ values of β-Lap in designed treatments. There is a negative association between mRNA levels of NQO1 and IC₅₀ values of β-Lap but not NQO2.     

Membrane interactions and the effect of metal ions of the amyloidogenic fragment Aβ(25-35) in comparison to Aβ(1-42)

Aβ(1−42) peptide, found as aggregated species in Alzheimer's disease brain, is linked to the onset of Alzheimer's disease. Many reports have linked metals to inducing Aβ aggregation and amyloid plaque formation. Aβ(25-35), a fragment from the C-terminal end of Aβ(1−42), lacks the metal coordinating sites found in the full-length peptide and is neurotoxic to cortical cortex cell cultures. We report solid-state NMR studies of Aβ(25-35) in model lipid membrane systems of anionic phospholipids and cholesterol, and compare structural changes to those of Aβ(1-42). When added after vesicle formation, Aβ(25-35) was found to interact with the lipid headgroups and slightly perturb the lipid acyl-chain region; when Aβ(25-35) was included during vesicle formation, it inserted deeper into the bilayer. While Aβ(25-35) retained the same β-sheet structure irrespective of the mode of addition, the longer Aβ(1-42) appeared to have an increase in β-sheet structure at the C-terminus when added to phospholipid liposomes after vesicle formation. Since the Aβ(25-35) fragment is also neurotoxic, the full-length peptide may have more than one pathway for toxicity.     

Unveiling Amyloid-β<Subscript>1–42</Subscript> Interaction with Zinc in Water and Mixed Hexafluoroisopropanol Solution in Alzheimer’s Disease

Alzheimer’s disease (AD) is a neurodegenerative disorder caused by overproduction and accumulation of amyloid beta-peptide (Aβ). The hallmarks associated with this AD are the presence of Aβ plaques between the nerve cell in the brain which leading to synaptic loss in memory. The amyloid plaques contain of transition metals like zinc, copper and iron. In a healthy brain, the metal ions are present in balance concentration. High concentrations of Zn are normally released during neurotransmission process. The release of Zn might cause the aggregation of Aβ leading to AD. Amyloid-β_1–42 is the main type of Aβ in amyloid plaque. There still have limited explanation on how Aβ_1–42 interaction with Zn metal, as well as the effect of Zn metal on the Aβ structure in different solvents in atomic detail. Therefore, we investigated the structural changes of Aβ_1–42 in water (Aβ-H₂O) and the mixed hexafluoroisopropanol (HFIP) with water (Aβ-HFIP/H₂O). The mixed solvent consisted of hexafluoroisopropanol (HFIP) and water was used with the ratio of HFIP:H₂O (80:20). The effect of zinc ion was also examined for the interaction of Aβ peptide with zinc in water (Aβ-Zn-H₂O) and mixed solvent (Aβ-Zn-HFIP/H₂O) using all atom level molecular dynamics (MD) calculations for 1 μs. We found that Aβ-Zn-HFIP/H₂O contained more α-helix compared to Aβ-HFIP/H₂O while Aβ-H₂O and Aβ-Zn-H₂O produced well-dissolved structure and they contained more β-sheets. β-turns are possible to bind with the receptor proteins and may induce the aggregation process in AD. Thus, Aβ-H₂O and Aβ-Zn-H₂O have higher possibility leading to AD compared to Aβ-Zn-HFIP/H₂O and Aβ-HFIP/H₂O models.