乙酰胆碱酯酶(AChE)与β淀粉样肽(Aβ)的相互关系

老年性痴呆症(Alzheimer‘s Disease,AD)是一种慢性的、进行性的神经系统退行性疾病。主要特征为各方面智力的损伤,包括学习记忆、语言、读写、行为,以及对周围环境的识别,最终可导致死亡。它有三大病理特征：(1)主要由β淀粉样肽(β—amyloid peptide,Aβ)沉积而成的淀粉样斑;(2)由高度磷酸化的Tau蛋白组成的神经纤维缠结;(3)神经元及神经突触的丢失。在AD疾病的发病机制及治疗的对策中,AD和乙酰胆碱酯酶(acetylcholinesterase,AChE)都有重要的作用。近年来对Aβ和AChE在AD疾病发生发展过程中相互作用的研究有了越来越多的报道。在此,作者对Aβ和AChE的关系作一综述。     

Cystine-knot peptides engineered with specificities for α(IIb)β(3) or α(IIb)β(3) and α(v)β(3) integrins are potent inhibitors of platelet aggregation

A truncated form of the Agouti‐related protein (AgRP), a member of the cystine‐knot family, has shown promise as a scaffold for engineering novel peptides with new molecular recognition properties. In this study, we replaced a constrained six amino acid loop in AgRP with a nine amino acid loop containing an Arg–Gly–Asp integrin recognition motif, and randomized the neighboring residues to create a library of ～20 million AgRP variants. We displayed the AgRP mutants as fusions on the surface of yeast and used high‐throughput fluorescence‐activated cell sorting (FACS) to isolate peptides that bound specifically to the platelet integrin α_IIbβ₃, a clinically important target for the prevention and treatment of thrombosis. These AgRP peptides had equilibrium dissociation (K_D) constants for α_IIbβ₃ integrin ranging from 60 to 90 nM, and did not bind to α_vβ₃, α_vβ₅, or α₅β₁ integrins. Using an alternate library screening strategy, we identified AgRP peptides that bound to both α_IIbβ₃ and α_vβ₃ integrins with K_D values ranging from 40 to 70 nM and 20 to 30 nM, respectively, and did not bind to α_vβ₅ or α₅β₁ integrins. Unique consensus sequences were identified within both series of AgRP peptides suggesting alternative molecular recognition events that dictate different integrin binding specificities. In addition, the engineered AgRP peptides prevented platelet aggregation as well as or slightly better than the FDA‐approved cyclic peptide eptifibatide. Collectively, these data demonstrate that cystine‐knot peptides can be generated with high affinity and specificity to closely‐related integrins, and provide insights into molecular interactions between small, structured peptide ligands and their receptors. Copyright © 2010 John Wiley & Sons, Ltd.     

Divalent copper ion bound amyloid-β(40) and amyloid-β(42) alloforms are less preferred than divalent zinc ion bound amyloid-β(40) and amyloid-β(42) alloforms

Divalent copper and zinc ions bind to the amyloid-β(40) and amyloid-β(42) alloforms and affect their structural stability as well as their chemical and physical properties. Current literature debates the impact of copper ions on amyloid-β alloforms. Recently, we reported the structural and thermodynamic properties of apo amyloid-β and divalent zinc ion bound amyloid-β alloforms (see, Wise-Scira et al. in J Biol Inorg Chem 17:927–938, 2012 and Coskuner et al. in ACS Chem Neurosci 4: 310–320, 2013). In our search for understanding the impacts of transition metal ions on disordered amyloid-β, we also developed and reported new potential functions using quantum mechanics, which are required for high-quality molecular dynamics simulations of divalent copper ion bound amyloid-β alloforms (see, Wise and Coskuner in J Comput Chem 35:1278–1289, 2014). The structures and thermodynamic properties of the divalent copper ion bound amyloid-β(40) and amyloid-β(42) alloforms in an aqueous medium are studied. The secondary and tertiary structures of divalent copper ion bound amyloid-β(40) and amyloid-β(42) along with their thermodynamic properties including enthalpy, entropy, Gibbs free energy and potential of mean force surface are investigated. Results are compared to those for apo amyloid-β and divalent zinc ion bound amyloid-β alloforms. Results demonstrate that copper binding to Aβ alloforms is thermodynamically less preferred rather than zinc binding. Less compact structures of copper ion bound amyloid-β alloforms possess reduced stability in comparison to zinc ion bound amyloid-β alloforms. Cu(II) binding impacts the thermodynamic properties, secondary and tertiary structural properties of Aβ40 and Aβ42.     

Antifouling poly(β-peptoid)s

A new type of polymer highly resistant to nonspecific protein adsorption is reported. Poly(N-methyl-β-alanine) (PMeA) and poly(N-ethyl-β-alanine) (PEtA) synthesized via cobalt-catalyzed carbonylative polymerization of N-methylaziridine and N-ethylaziridine were end-functionalized with thiol groups and grafted onto Au surfaces. Protein adsorption was studied by the surface plasmon resonance (SPR) method. The amounts of representative single proteins adsorbed onto the PMeA- and PEtA-grafted surfaces were below the detection limit of SPR at the pg/mm(2) level. After exposure to full blood plasma and serum for 10 min, protein adsorption was at the level of ～ 100 pg/mm(2), similar to the level of protein adsorption on poly(ethylene glycol) surfaces subjected to identical conditions. These poly(β-peptoid)s therefore provide excellent protein resistance comparable to the best antifouling materials known to date. The strong proton-accepting ability when forming hydrogen bonds is suggested to be an important attribute for these poly(β-peptoid)s as well as other poly(tertiary amide)s as antifouling materials.     

β-Diiminatolanthanoid(III) halides revisited

The crystalline compounds [LnCl₂(L)(thf)₂] [Ln = Ce (1), Tb (2), Yb (3)], [NdI₂(L)(thf)₂] (4), [LnCl(L′)₂] [Ln = Tb (5), Yb (6) (a known compound)] and [YbCl(L′′)(μ-Cl)₂Li(OEt₂)₂] (7) have been prepared [L = {N(C₆H₃Prⁱ₂-2,6)C(H)}₂CPh, L′ = {N(SiMe₃)C(Ph)}₂CH, L′′ = {N(SiMe₃)C(C₆H₄Ph-4)}₂CH]. The X-ray molecular structures of 2-7 have been established; in each, the monoanionic ligand L, L′ or L′′ is N,N′-chelating and essentially π-delocalised. Each of 1-7 was prepared from the appropriate LnCl₃, or for 4 [NdI₃(thf)₂], and an equivalent portion of the appropriate alkali metal [Li for 7, Na for 2, 3 and 5, or K for 1, 4 and 6] β-diiminate in thf; the isolation of exclusively 5 and 6 (rather than the L′ analogues of 2 or 3) is noteworthy, as is the structure of 7 which has no precedent in Group 3 or 4f metal β-diiminato chemistry.     

β-内酰胺酶(β-Lactamase)抑制剂的抑菌试验

自从抗生素被发现以来,许多传染病都得到了控制。然而随着抗生素的广泛应用,尤其是不合理的滥用,微生物对抗生素的耐药性问题也越趋严重,耐青霉素的细菌已达90％。对广泛使用的β-内酰胺类抗生素(青霉素、头孢霉素)的主要耐     

β-Hydroxy-β-methylbutyrate (HMB) prevents dexamethasone-induced myotube atrophy

High levels of glucocorticoids result in muscle wasting and weakness. β-hydroxy-β-methylbutyrate (HMB) attenuates the loss of muscle mass in various catabolic conditions but the influence of HMB on glucocorticoid-induced muscle atrophy is not known. We tested the hypothesis that HMB prevents dexamethasone-induced atrophy in cultured myotubes. Treatment of cultured L6 myotubes with dexamethasone resulted in increased protein degradation and expression of atrogin-1 and MuRF1, decreased protein synthesis and reduced myotube size. All of these effects of dexamethasone were attenuated by HMB. Additional experiments provided evidence that the inhibitory effects of HMB on dexamethasone-induced increase in protein degradation and decrease in protein synthesis were regulated by p38/MAPK- and PI3K/Akt-dependent cell signaling, respectively. The present results suggest that glucocorticoid-induced muscle wasting can be prevented by HMB.     

β-β交联的Fe(Ⅱ)-Co(Ⅱ)杂化血红蛋白玻尔(Bohr)效应的研究

本文使用500MHz ~1H NMR谱仪研究了β—β交联的β_1 82Lys—β_2 82Lys不对称Fe(Ⅱ)Co(Ⅱ)杂化血红蛋白在去氧状态下其四级结构与溶液 pH的相互关系.实验结果证明,溶液中H~+浓度的减少,有利于血红蛋白从束缚态(T态)向松弛态(R态)方向转化.在可交换质子区(9.0—15.0PPm)由pH的改变所引起的[2(Co)β(Fe)]_A[α(Co)β(Co)]_cXL的~1H NMR谱峰的变化比[α(Fe)β(Co)]_A[α(Co)β(Co)]_cXL的~1H NMR谱峰的变化大.这为研究血红蛋白四个亚基的构象随pH改变而变化的先后次序提供了一定的依据,可以认为,当溶液中H~+浓度减少时,首先使蛋白构象发生改变的应在α—亚基上,而不在β—亚基上.此外,从它们的~1H NMR谱图随pH的变化可推测,溶液中除了 T态和R态之外,还存在着一些中间过渡态.o     

Transforming growth factor-β1 (TGF-β1) and acetylcholine (ACh) alter nitric oxide (NO) and interleukin-1β (IL-1β) secretion in human colon adenocarcinoma cells

Colon adenocarcinoma is one of the most common fatal malignancies in Western countries. Progression of this cancer is dependent on tumor microenvironmental signaling molecules such as transforming growth factor-β (TGF-β) or acetylcholine (ACh). The present study was conducted to assess the influence of recombinant human transforming growth factor (rhTGF)-β1 or ACh on nitric oxide (NO) and interleukin-1β (IL-1β) secretion by three human colon adenocarcinoma cell lines: HT29, LS180, and SW948, derived from different grade tumors (Duke’s stage). The cells were cultured in 2D and 3D (spheroids) conditions. Colon carcinoma cells exhibited different sensitivities to rhTGF-β1 or ACh dependent on the tumor grade and the culture model. ACh exhibited significant inhibitory effects towards NO, endothelial nitric oxide synthase (eNOS), and IL-1β secretion especially by tumor cells derived form Duke’s C stage of colon carcinoma. rhTGF-β1 also decreased NO, IL-1β, and eNOS expression, but its effect was lower than that observed after the administration of ACh. The inhibition of NO and IL-1β production was more striking in 3D tumor spheroids than in 2D culture monolayers. Taken together, the TGF-β1–ACh axis may regulate colon carcinoma progression and metastasis by altering NO secretion and influence inflammatory responses by modulating IL-1β production.     

Synthesis of 1-β-L-Arabinofuranosylcytosine (β-L-Ara-C) and 2′-Deoxy-2′-methylene-β-L-cytidine (β-L-DMDC) as Potential Antineoplastic Agents

Abstract

1-β-L-Arabinofuranosylcytosine (β-L-Ara-C, 7) and 2′-deoxy-2′-methylene-β-L-cytidine (β-L-DMDC, 14) have been synthesized via a multi-step synthesis from L-arabinose. These compounds were tested in vitro against L1210, P388, Sarcoma 180, and CEM cells, and found not to be active at a concentration up to 100 μM. β-L-Ara-C and β-L-DMDC were also tested against HSV-1 and HSV-2 and yielded ID₅₀ values of 100 μM.     

Regioselective allylation of cyclomaltoheptaose (β-cyclodextrin) leading to per(2,6-di-O-hydroxypropyl-3-O-methyl)-β-cyclodextrin

The selective modification of cyclodextrins remains a real challenge to obtain well-defined structures. The targeted cycloheptakis-(1→4)-2,6-di-O-hydroxypropyl-3-O-methyl-α-d-glucopyranosyl [per(2,6-di-O-hydroxypropyl-3-O-methyl)-β-CD] was obtained by a three-step procedure. The selective allylation of the hydroxyl functions at the positions 2 and 6 was used as a first step. This reaction was revisited then enlarged to α and γ-CDs to determine new conditions for a one-step synthesis in high yield. The per(2,6-di-O-allyl)-β-CD derivative was then reacted with iodomethane to provide per(2,6-di-O-allyl-3-O-methyl)-β-CD. Oxidative hydroboration of the allyl functions was then carried out in order to obtain a new CD derivative with seven primary hydroxyl functions on each side of the truncated cone, having a similar reactivity.     

人17β羟类固醇脱氢酶(17β-HSD)基因在家蚕系统中的表达(简报)

17β羟类固醇脱氢酶(17β-hydroxystero-id dehydrogenase,17β-HSD)能催化人体内类固醇性激素的形成和转化,例如它可以催化雌二醇与雌酮,雄烯二酮与睾酮之间的相互转化反应。人体内17β-HSD在胎盘组织含量最为丰富,催化17β-雌二醇等雌激素的生成,同时这些激素能够刺激乳腺瘤的增生。因此,如何抑制17β-HSD酶的过高活性,减少癌变发生的可能性,已成为目前这类癌症治疗的一个重要研究目标。目前,从胎盘组织中提取17β-HSD的方法,产量低,比活小,周期长,     

γ-Secretase complexes containing caspase-cleaved presenilin-1 increase intracellular Aβ(42) /Aβ(40) ratio

Markers for caspase activation and apoptosis have been shown in brains of Alzheimer's disease (AD) patients and AD-mouse models. In neurons, caspase activation is associated with elevated amyloid β-peptide (Aβ) production. Caspases cleave numerous substrates including presenilin-1 (PS1). The cleavage takes place in the large cytosolic loop of PS1-C-terminal fragment (PS1CTF), generating a truncated PS1CTF lacking half of the loop domain (caspCTF). The loop has been shown to possess important regulatory functions with regard to Aβ(40) and Aβ(42) production. Previously, we have demonstrated that γ-secretase complexes are active during apoptosis regardless of caspase cleavage in the PS1CTF-loop. Here, a PS1/PS2-knockout mouse blastocyst-derived cell line was used to establish stable or transient cell lines expressing either caspCTF or full-length CTF (wtCTF). We show that caspCTF restores γ-secretase activity and forms active γ-secretase complexes together with Nicastrin, Pen-2, Aph-1 and PS1-N-terminal fragment. Further, caspCTF containing γ-secretase complexes have a sustained capacity to cleave amyloid precursor protein (APP) and Notch, generating APP and Notch intracellular domain, respectively. However, when compared to wtCTF cells, caspCTF cells exhibit increased intracellular production of Aβ(42) accompanied by increased intracellular Aβ(42) /Aβ(40) ratio without changing the Aβ secretion pattern. Similarly, induction of apoptosis in wtCTF cells generate a similar shift in intracellular Aβ pattern with increased Aβ(42) /Aβ(40) ratio. In summary, we show that caspase cleavage of PS1 generates a γ-secretase complex that increases the intracellular Aβ(42) /Aβ(40) ratio. This can have implications for AD pathogenesis and suggests caspase inhibitors as potential therapeutic agents.     

Penicillinase (β-lactamase) formation by blue-green algae

-Lactamase (penicillinase) activity was found in a number of strains of blue-green algae. In some cases, this enzyme permitted algae to overcome the inhibitory effects of penicillin. Production and localization of -lactamase were studied in a unicellular species, Coccochloris elabens (strain 7003), and in a filamentous, nitrogen-fixing Anabaena species (strain 7120). When cells were grown in a neutral medium with NaNO₃ as N source, the pH rose during growth; at a pH of about 10, most of the enzyme was extracellular and all the cell-bound enzyme was expressed equally well in intact or disrupted cells. If the pH was kept near neutrality during growth by gassing with CO₂ in N₂ or by growth under conditions of N₂ fixation, the enzyme remained cell-bound and cryptic for most of the growth phase, being measurable only after cells were disrupted. The enzymes from strains 7003 and 7120 had greater activity on benzyl penicillin and other penicillins than on cephalo-sporins. Some differences were observed in the substrate profiles of penicillinases from the two strains against different penicillins.A preliminary account of this work was presented at the 1974 meetings of the American Society for Microbiology in Chicago (Abstracts of Meetings, M37)     

β-Amylolytic activities ofEmericella nidulans (eidam) vuill

Summary Screening of fungal isolates led to selection of a strain ofEmericella nidulans 45 producing exocellular -amylase in a starch medium. Studies of dialysed enzyme and the formulation of the medium for the enzyme production are presented.     

Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.)

Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP[U-¹⁴C]glucose, the main components of which were identified as (1,3)(1,4)-- and (1,3)--D-glucans. The membrane preparations also catalysed the transformation of UDP-glucose into labelled low-molecular-weight products, mainly glucose (by phosphatase action), glucose-1-phosphate (by phosphodiesterase action) and glyco(phospho)lipids (by glycosyltransferase action). The formation of (1,3)(1,4)--glucans, (1,3)--glucans, and the other reactions competing for UDP-glucose, were monitored simultaneously and quantitatively by a novel procedure based on enzymatic analysis, thin-layer chromatography and digital autoradiography. Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)--glucan synthesis or (1,3)--glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4) and (1,3)--glucan synthesis reactions even when both occurred together. The synthesis of both -glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)--glucan synthesis and (1,3)--glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg²⁺: (1,3)--glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)--glucan synthesis continued to increase up to 200 mM Mg²⁺, when the ion was supplied as the sulphate. (1,3)--Glucan synthesis was Ca²⁺ dependent and this dependence could be abolished by proteinase treatment. The K _m with respect to UDP-glucose was 1.5 mM for (1,3)--glucan synthesis and approximately 1 mM for (1,3)(1,4)--glucan synthesis. The (1,3)(1,4)--glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)--glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.Abbreviations G4G4G3G Glc(1,4)Glc(1,4)Glc(1,3)Glc (-linked) - UDP-Glc uridine-5-diphosphate glucose We are grateful to the Commission of the European Communities for the award of Training Fellowships to Christine Vincent and Martin Becker.     

Preparation of 9-β-D-Arabinofuranosylguanine (araG)

Abstract

A convenient practical synthesis of araG is described.     

Structural diversity of neutral bis-(β-iminoaldehyde) complexes of nickel(II)

Series of Ni^II and Cu^II complexes with dianionic [N₂O₂] ligands were synthesized and characterised applying spectroscopic and X-ray diffraction techniques. The ligands were obtained by 1:2 condensation of ethylene- and propylenediamine with malonic aldehyde derivatives (R² = H, R¹ = H or OCH₃). Although the molecular formulae of the complexes are quite similar, the X-ray investigations have proved a significant structural diversity in the solid state. Among others, we found some simple nearly planar molecules stacked in the crystal lattice with electron density of six-membered rings delocalised over the chelate rings as well as some very complex polymeric or nickel acetate bridged trinuclear complexes. The coordination of the nickel ion by surrounding oxygen and nitrogen atoms is square-planar in the simplest case and octahedral in the most complex one. Small topological differences in similar molecules generate completely different crystal structures.From magnetic studies, a small, negative value of J obtained confirms the occurrence of weak antiferromagnetic interactions between the Ni^II ions in polymeric chain of the propylenediamine dialdehyde substituted derivative.     

Development and validation of competition binding assays for affinity to the extracellular matrix receptors, α(v)β(3) and α(IIb)β(3) integrin

The RGD (Arg-Gly-Asp) binding integrins α(v)β(3) and α(IIb)β(3) are integral components of various pathological and physiological processes, including tumor angiogenesis, osteoclast function, and thrombus formation. Because of this, there is interest in identifying novel compounds and proteins binding to these receptors as well as investigating the mechanism of these interactions. In this article, we describe the development and validation of competition binding assays for determining the affinity of test compounds to α(v)β(3) and α(IIb)β(3) integrin. Assays were successfully developed for each receptor, and the affinity of known compounds was comparable to published results. However, the inability of binding between α(IIb)β(3) integrin and the labeled echistatin protein ligand to reach equilibrium resulted in an assay that did not meet the assumptions of the competition binding model. Nevertheless, there was good agreement between this assay and known literature values, and intra- and interassay variability was acceptable. Binding by conformation-specific antibodies provided evidence that solid-phase bound α(IIb)β(3) receptor was in an activated conformation. This study also demonstrated that current models and methods for determining receptor affinity are simplistic and fail to account for common receptor-ligand interactions such as nondissociable interactions and varying receptor activation states.