Inhibition of cytotoxicity and amyloid fibril formation by a D-amino acid peptide that specifically binds to Alzheimer's disease amyloid peptide

Alzheimer's disease (AD) is a progressive neurodegenerative disorder. The 'amyloid cascade hypothesis' assigns the amyloid-beta-peptide (Abeta) a central role in the pathogenesis of AD. Although it is not yet established, whether the resulting Abeta aggregates are the causative agent or just a result of the disease progression, polymerization of Abeta has been identified as a major feature during AD pathogenesis. Inhibition of the Abeta polymer formation, thus, has emerged as a potential therapeutic approach. In this context, we identified peptides consisting of d-enantiomeric amino acid peptides (d-peptides) that bind to Abeta. D-peptides are known to be more protease resistant and less immunogenic than the respective L-enantiomers. Previously, we have shown that a 12mer D-peptide specifically binds to Abeta amyloid plaques in brain tissue sections from former AD patients. In vitro obtained binding affinities to synthetic Abeta revealed a K(d) value in the submicromolar range. The aim of the present study was to investigate the influence of this d-peptide to Abeta polymerization and toxicity. Using cell toxicity assays, thioflavin fluorescence, fluorescence correlation spectroscopy and electron microscopy, we found a significant effect of the d-peptide on both. Presence of D-peptides (dpep) reduces the average size of Abeta aggregates, but increases their number. In addition, Abeta cytotoxicity on PC12 cells is reduced in the presence of dpep.     

A standardized and biocompatible preparation of aggregate-free amyloid beta peptide for biophysical and biological studies of Alzheimer's disease

We provide a validated and rapid protocol for the solubilization of amyloid β-peptide (Aβ). This procedure involves sequential solubilization using structure-breaking organic solvents hexafluoroisopropanol and DMSO followed by column purification. The low solubility and tendency of Aβ to aggregate considerably impede the in vitro handling and biophysical or biological investigation of Aβ, despite the interest in this peptide because of its implication in Alzheimer's disease. The main advantage of the proposed protocol over others is that it results in standardized aggregate-free Aβ peptide samples that are biocompatible for cell culture studies and yield reproducible aggregation kinetics and cytotoxicities. This three-step protocol also enables the co-solubilization of the longer Aβ42 variant with Aβ40 in ratios relevant to Alzheimer's disease.     

Selection of D-amino-acid peptides that bind to Alzheimer's disease amyloid peptide abeta1-42 by mirror image phage display

A mirror image phage display approach was used to identify novel and highly specific ligands for Alzheimer's disease amyloid peptide Abeta(1-42). A randomized 12-mer peptide library presented on M13 phages was screened for peptides with binding affinity for the mirror image of Abeta(1-42). After four rounds of selection and amplification the peptides were enriched with a dominating consensus sequence. The mirror image of the most representative peptide (D-pep) was shown to bind Abeta(1-42) with a dissociation constant in the submicromolar range. Furthermore, in brain tissue sections derived from patients that suffered from Alzheimer's disease, amyloid plaques and leptomeningeal vessels containing Abeta amyloid were stained specifically with a fluorescence-labeled derivative of D-pep. Fibrillar deposits derived from other amyloidosis were not labeled by D-pep. Possible applications of this novel and highly specific Abeta ligand in diagnosis and therapy of Alzheimer's disease are discussed.     

水泥厂主要有害气体及其防治

叙述了水泥生产过程中产生的主要有害气体,论述了它们的理化特征及给人类社会带来的危害。有害气体的防治技术很多,但有些成本很高,有的尚未进入实用阶段。文章结合我国国情,针对《水泥工业大气污染物排放标准》GB4915-2004的规定,重点对二氧化硫、氮氧化物等气态污染物的降排处理,介绍了一些较为简单有效而又适用的防治技术。另外,对产生温室效应的罪魁——二氧化碳的产生机理、数量及减排措施也提出了见解,并建议不失时机地利用清洁发展机制（CDM）来获得资金援助和先进技术。     

The slowly aggregating salmon Calcitonin: a useful tool for the study of the amyloid oligomers structure and activity

Amyloid proteins of different aminoacidic composition share the tendency to misfold and aggregate in a similar way, following common aggregation steps. The process includes the formation of dimers, trimers, and low molecular weight prefibrillar oligomers, characterized by the typical morphology of globules less than 10 nm diameter. The globules spontaneously form linear or annular structures and, eventually, mature fibers. The rate of this process depends on characteristics intrinsic to the different proteins and to environmental conditions (i.e., pH, ionic strength, solvent composition, temperature). In the case of neurodegenerative diseases, it is now generally agreed that the pathogenic aggregates are not the mature fibrils, but the intermediate, soluble oligomers. However, the molecular mechanism by which these oligomers trigger neuronal damage is still unclear. In particular, it is not clear if there is a peculiar structure at the basis of the neurotoxic effect and how this structure interacts with neurons. This review will focus on the results we obtained using salmon Calcitonin, an amyloid protein characterized by a very slow aggregation rate, which allowed us to closely monitor the aggregation process. We used it as a tool to investigate the characteristics of amyloid oligomers formation and their interactions with neuronal cells. Our results indicate that small globules of about 6 nm could be the responsible for the neurotoxic effects. Moreover, our data suggest that the rich content in lipid rafts of neuronal cell plasma membrane may render neurons particularly vulnerable to the amyloid protein toxic effect.     

Individually randomized intervention trials for disease prevention and control

It is argued that randomized, controlled trials should fulfil a critical role in the identification of practical approaches to the prevention and control of chronic diseases. Because of the great public health potential of chemopreventive and behavioural approaches to chronic disease prevention there is need for a major interdisciplinary scientific effort aimed at intervention development. Because of the cost and duration of controlled trials to evaluate specific interventions there is a need for well-conducted feasibility, pilot and intermediate outcome trials, to inform and to justify corresponding full-scale trials having clinical disease outcomes. Compared to therapeutic trials, prevention trials need to have a greater emphasis on overall benefit versus risk assessment. Such trials need to be large enough, and of sufficient duration, to yield powerful tests of key hypotheses, and informative benefit versus risk summary statements. These requirements have a range of implications for intervention trial design, conduct, monitoring and reporting, which are reviewed and discussed. The clinical trial component of the ongoing Women's Health Initiative provides illustration throughout this discussion.     

The biochemical and cellular basis for nutraceutical strategies to attenuate neurodegeneration in Parkinson's disease

Future therapeutic intervention that could effectively decelerate the rate of degeneration within the substantia nigra pars compacta (SNc) could add years of mobility and reduce morbidity associated with Parkinson's disease (PD). Neurodegenerative decline associated with PD is distinguished by extensive damage to SNc dopaminergic (DAergic) neurons and decay of the striatal tract. While genetic mutations or environmental toxins can precipitate pathology, progressive degenerative succession involves a gradual decline in DA neurotransmission/synaptic uptake, impaired oxidative glucose consumption, a rise in striatal lactate and chronic inflammation. Nutraceuticals play a fundamental role in energy metabolism and signaling transduction pathways that control neurotransmission and inflammation. However, the use of nutritional supplements to slow the progression of PD has met with considerable challenge and has thus far proven unsuccessful. This review re-examines precipitating factors and insults involved in PD and how nutraceuticals can affect each of these biological targets. Discussed are disease dynamics (Sections 1 and 2) and natural substances, vitamins and minerals that could impact disease processes (Section 3). Topics include nutritional influences on α-synuclein aggregation, ubiquitin proteasome function, mTOR signaling/lysosomal-autophagy, energy failure, faulty catecholamine trafficking, DA oxidation, synthesis of toxic DA-quinones, o-semiquinones, benzothiazolines, hyperhomocyseinemia, methylation, inflammation and irreversible oxidation of neuromelanin. In summary, it is clear that future research will be required to consider the multi-faceted nature of this disease and re-examine how and why the use of nutritional multi-vitamin-mineral and plant-based combinations could be used to slow the progression of PD, if possible.     

In silico theoretical molecular modeling for Alzheimer's disease: the nicotine-curcumin paradigm in neuroprotection and neurotherapy

The aggregation of the amyloid-β-peptide (AβP) into well-ordered fibrils has been considered as the key pathological marker of Alzheimer's disease. Molecular attributes related to the specific binding interactions, covalently and non-covalently, of a library of compounds targeting of conformational scaffolds were computed employing static lattice atomistic simulations and array constructions. A combinatorial approach using isobolographic analysis was stochastically modeled employing Artificial Neural Networks and a Design of Experiments approach, namely an orthogonal Face-Centered Central Composite Design for small molecules, such as curcumin and glycosylated nornicotine exhibiting concentration-dependent behavior on modulating AβP aggregation and oligomerization. This work provides a mathematical and in silico approach that constitutes a new frontier in providing neuroscientists with a template for in vitro and in vivo experimentation. In future this could potentially allow neuroscientists to adopt this in silico approach for the development of novel therapeutic interventions in the neuroprotection and neurotherapy of Alzheimer's disease. In addition, the neuroprotective entities identified in this study may also be valuable in this regard.     

O型口蹄疫病毒血清抗体竞争ELISA检测方法的建立及验证

目的建立O型口蹄疫病毒(foot-and-mouth disease virus,FMDV)血清抗体的竞争ELISA检测方法,并进行验证。方法采用O型FMDV病毒样颗粒(virus-like particle,VLP)作为包被抗原,建立用于检测FMDV血清抗体的竞争ELISA方法。对方法的抗原包被浓度(0. 1、0. 2、0. 3、0. 4、0. 5、0. 6、0. 7、0. 8、0. 9、1. 0μg/mL)、抗原包被温度及时间(4℃过夜、37℃1 h、37℃1.5 h、37℃2 h、37℃2. 5 h、37℃3 h)、血清稀释度(1∶2、1∶4、1∶8、1∶16稀释)、HRP-IgG稀释度(1∶10 000～1∶20 000稀释)、封闭液种类(不封闭、1%BSA封闭、2%BSA封闭、5%脱脂乳封闭)、封闭液作用时间(30、60、90、120 min)、血清与HRP-IgG作用时间(30、60、90、120、150 min)、底物TMB作用时间(10、15、20、25、30 min)进行优化。同时验证方法的交叉反应、特异性及敏感性、精密性。采用建立的方法及O型FMDV抗体液相阻断EL...     

Novel Pyridine-Containing Sultones: Structure-Activity Relationship and Biological Evaluation as Selective AChE Inhibitors for the Treatment of Alzheimer's disease

Novel pyridine-containing sultones were synthesized and evaluated for their cholinesterase (ChE) inhibitory activity. Most of compounds showed selective acetylcholinesterase (AChE) inhibitory activity. The structure-activity relationship (SAR) showed: (i) the fused pyridine-containing sultones increase AChE inhibition, series B >series A ; (ii) for series A , the effect of the 4-substituent on AChE activity, p->m- or o-; (iii) for series B , a halophenyl group increase activity. Compound B4 (4-(4-chlorophenyl)-2,2-dioxide-3,4,5,6-tetrahydro-1,2-oxathiino[5,6-h]quinoline) was identified as a selective AChE inhibitor (IC₅₀=8.93 μM), and molecular docking studies revealed a good fit into TcAChE via hydrogen interactions between the δ-pyridylsultone scaffold with Asp72, Ser122, Phe288, Phe290 and Trp84. Compound B4 showed reversible and non-competitive (K_i=7.67 μM) AChE inhibition, nontoxicity and neuroprotective activity. In vivo studies confirmed that compound B4 could ameliorate the cognitive performance of scopolamine-treated C57BL/6 J mice, suggesting a significant benefit of AChE inhibition for a disease-modifying treatment of AD.     

Synthesis and pharmacological evaluation of 8- and 9-substituted benzolactam-v8 derivatives as potent ligands for protein kinase C, a therapeutic target for Alzheimer's disease

A central element in the pathophysiology of Alzheimer's disease (AD) is the formation of amyloid plaques, which result from abnormal processing of the amyloid precursor protein (APP). The processing of APP is largely provided by three key enzymes, namely the alpha-, beta-, and gamma-secretases. As the latter two contribute to the formation of neurotoxic Abeta fragments while alpha-secretase does not, a decrease in the amyloidogenic products can be brought about either by inhibition of the beta- and gamma-secretases or through the activation of alpha-secretase. It is now known that the activation of protein kinase C (PKC) enhances alpha-secretase activity and therefore represents a possible target for the development of agents urgently needed for the treatment of this devastating neurodegenerative disorder. In the present study, new benzolactam-V8-based PKC activators were synthesized and tested for their binding affinity toward PKCalpha. All compounds tested showed binding values in the nanomolar concentration range. In accordance with previous publications, 9-substitution dramatically increased PKC binding affinity in comparison with the corresponding 8-substituted analogues. In addition to the location of the side chain on the aromatic ring, the binding affinities of these benzolactams were found to depend on the orientation, length, and electronic properties of this appendage. An interesting decrease in binding affinity was found for the 9-thienyl analogue 13, suggesting adverse electronic interactions of the sulfur atom with PKC or parts of the cellular membrane.     

Designing and operating a pilot plant for purification of industrial wastewater from toxic organic compounds by utilizing solar energy

The aim of the present project was to design and operate a solar reactor system and to analyze its performance for the removal of different types of toxic organic pollutants (e.g., synthetic methyl violet dye and phenol) from water with titanium dioxide as the photocatalyst. Various operating parameters were studied to investigate the behavior of the designed reactor like initial substrate concentration, loading of catalyst, pH of solution, and H₂O₂ concentration. The operating parameters were optimized to give higher efficiency to the reactor performance. Results showed that a photocatalysis system, operating at optimum conditions, offered within one hour of operation degradation up to 95.27% for synthetic dye, while a conversion of 99.95% was obtained in three hours. With phenol, degradation was up to 80.0% and 98.0%, respectively. The removal of TOC for the two toxic materials was also at high levels. This confirmed the feasibility of the designed solar system. The kinetics of dye degradation was first order with respect to dye concentration and could be well described by Langmuir-Hinshelwood model. A preliminary design of a solar photocatalysis system as an alternative treatment method for wastewater effluents from an Iraqi textile mill was introduced.     

重组戊型肝炎p179疫苗小鼠抗体间接ELISA检测方法的建立及其初步应用

目的建立重组戊型肝炎p179疫苗抗体间接ELISA检测方法,并进行初步应用。方法确定重组戊型肝炎疫苗p179抗原的最佳包被浓度及包被条件、酶标抗体的最佳稀释度、封闭条件、最佳抗体稀释液,对建立的方法的特异性、准确性及精密度进行验证,并与市售ELISA试剂盒进行比较。结果抗原最佳包被浓度为1μg/ml,4℃包被过夜;酶标抗体最佳稀释度为1∶5 000;3%牛血清白蛋白封闭3 h;最佳抗体稀释液为含1%酪蛋白PBS。建立的方法检测的A450与重组戊肝抗原浓度呈明显的剂量依赖性,对不相关的anti-HAV、anti-HB、anti-INF、anti-RABV无交叉反应;36份小鼠阳性血清的检出率为100%;3份阳性血清重复检测20次的CV均小于15%。市售某厂家ELISA试剂盒检测48份血清,阳性检出率为83.3%,建立的间接ELISA方法检测,阳性检出率为89.6%。结论建立了重组戊肝p179疫苗抗体间接ELISA检测方法,可用于重组戊肝疫苗小鼠免疫后血清抗体的检测。     

流感减毒活疫苗诱发黏膜IgA抗体间接ELISA检测方法的优化及其实验室内控品的制备

目的优化流感减毒活疫苗诱发黏膜IgA抗体的ELISA检测方法,并制备IgA抗体实验室内控品。方法采用ELISA检测法对流感病毒抗原(全病毒及其相应HA抗原)及包被浓度(0. 5、1、2、4μg/mL)、封闭液(1%BSA、10%FBS、5%脱脂奶粉)及封闭时间(1、1. 5、2 h)、样本稀释液(含1%BSA的PBST、含2%脱脂奶粉的PBST、样本保存液)及样本作用时间(45、60、75 min)、酶标抗体稀释度(1∶1 000、1∶2 000、1∶4 000、1∶8 000)进行优化。采用优化的方法制备针对H1N1、H3N2和B型3个型别流感病毒的IgA抗体检测用实验内控品,并用该方法对接种三价流感减毒活疫苗的20名志愿者的鼻咽拭子样本进行检测。结果最适间接ELISA法检测条件为:以全病毒作为包被抗原,包被浓度为1μg/mL;以含1%牛血清白蛋白为封闭液封闭2 h;样本稀释液为2%脱脂奶粉的PBST,作用时间为60 min;酶标抗体稀释度为1∶1 000。采用优化方法制备的H1N1、H3N2、B型IgA抗体阳性实验室内控品几何平均滴度分别为23、45、35,可接受范围分别为16～32、32～64、32～64。接种疫苗后20名志愿者中3种型别IgA抗体滴度较接种前均显著增高(P 0. 001),40%～50%志愿者接种后IgA抗体滴度比接种前至少升高2倍。结论成功优化了流感减毒活疫苗诱发黏膜IgA抗体的ELISA检测方法,制备的IgA抗体的实验室内控品可用于流感减毒活疫苗接种后鼻咽拭子抗体的检测。     

Distance measurement between Tyr10 and Met35 in amyloid beta by site-directed spin-labeling ESR spectroscopy: implications for the stronger neurotoxicity of Abeta42 than Abeta40

The neurotoxicity of the 42-mer and 40-mer amyloid beta peptides (Abeta42 and Abeta40) is closely related to the radicalization at both Tyr10 and Met35. Abeta42 is more neurotoxic than Abeta40. Our previous structural analyses of Abeta42 suggested that Tyr10 and Met35 are brought closer together by the turn at positions 22 and 23, and the S-oxidized radical cation at position 35, which is the ultimate toxic radical species, can be produced effectively through oxidation by the phenoxy radical at position 10. To verify this idea, their separation was measured by site-directed spin labeling (MTSSL) by using ESR spectroscopy. Among the three kinds of Abeta42 derivatives, which are doubly or singly spin-labeled at position 10 and 35, only 10,35-MTSSL-Abeta42 showed a clear dipole coupling in continuous-wave ESR; this suggests that the intramolecular spin labels at position 10 and 35 in Abeta42 are located within approximately 15 A. In contrast, 10,35-MTSSL-Abeta40 did not give such signals. The distance between Tyr10 and Met35 in 10,35-MTSSL-Abeta40, which was successfully measured by pulsed ESR spectroscopy was 30 A long. The difference in the distance between Abeta42 and Abeta40 could explain in part the stronger neurotoxicity of Abeta42 compared to Abeta40.     

前蛋白转化酶枯草溶菌素9单克隆抗体生物学活性荧光染料标记法的建立、优化及验证

目的建立并优化前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin/kexin type 9,PCSK9)单克隆抗体生物学活性的荧光染料标记检测法,并进行验证。方法对建立的荧光染料标记法的细胞铺板密度(1×10~5、5×10~5和1×10~6个/mL)、PCSK9蛋白浓度(5、20、40、80μg/mL)、荧光标记的低密度脂蛋白(low density lipoprotein labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate,Dil-LDL)浓度(10、16、20μg/mL)、单克隆抗体的浓度梯度(200、52、20、12、8、4、0. 8μg/mL,200、30、10、6、4、1、0. 2μg/mL,200、28、9、6、4、2、0. 2μg/mL)进行优化,并对优化方法的专属性、准确度、精密度、线性范围、耐用性进行验证。结果最适方法检测条件:铺板密度为5×10~5个/mL,PCSK9蛋白和Dil-LDL浓度分别为20和16μg/mL,单克隆抗体的浓度梯度为200、52、20、12、8、4、0. 8μg/mL。该方法可特异性检测PCSK9单克隆抗体;供试品检测回收率在70%～130%之间;供试品检测结果的相对标准偏差(RSD)均10%;供试品效价在50%～150%之间时,检测值与理论值呈良好的线性关系,线性拟合方程为y=1. 002 x-0. 006 7,R~2=0. 997 8;耐用性检测活性均在70%～140%范围内。结论本实验建立的方法具有良好的专属性、准确度和精密度,可用于PCSK9单克隆抗体的生物学活性测定。     

Optimal design of the coat‐hanger die used for producing melt‐blown fabrics by finite element method and evolution strategies

In this article, an optimal design procedure that improves the uniformity of flow rate distribution at the outlet of the coat‐hanger die is proposed. The two‐membered evolution strategy was combined with the finite element method to optimize the design parameters of an initial coat‐hanger die geometry designed by analytical method based on one‐dimensional lubrication method. The slot gap and the manifold angle were chosen to be the optimized design parameters, and the coefficient of variation (CV) value of the flow velocity at the die outlet is regarded as the objective function. The optimal results were achieved in the 22nd generation after 100 generations' evolution, which show that the CV% value of the flow velocity at the die outlet is only 1.3631% and decreases by 68% of the initial value caused by unoptimizable die geometry. POLYM. ENG. SCI., 2009. © 2008 Society of Plastics Engineers