Polymer-Based Module for NAD+ Regeneration with Visible Light

The regeneration of enzymatic cofactors by cell-free synthetic modules is a key step towards producing a purely synthetic cell. Herein, we demonstrate the regeneration of the enzyme cofactor NAD⁺ by photo-oxidation of NADH under visible-light irradiation by using metal-free conjugated polymer nanoparticles. Encapsulation of the light-active nanoparticles in the lumen of polymeric vesicles produced a fully organic module able to regenerate NAD⁺ in an enzyme-free system. The polymer compartment conferred physical and chemical autonomy to the module, allowing the regeneration of NAD⁺ to occur efficiently, even in harsh chemical environments. Moreover, we show that regeneration of NAD⁺ by the photocatalyst nanoparticles can oxidize a model substrate, in conjunction with the enzyme glycerol dehydrogenase. To ensure the longevity of the enzyme, we immobilized it within a protective silica matrix; this yielded enzymatic silica nanoparticles with enhanced long-term performance and compatibility with the NAD⁺-regeneration system.     

高温液态水中烟腈水解反应动力学

为了开发烟酸和烟酰胺的绿色制备方法以及探讨高温液态水中腈类物质的水解规律,研究了高温液态水中烟腈的无催化水解反应动力学.考察了实验数据的重现性以及烟腈初始质量浓度对反应动力学的影响,测定了48315～52315 K下烟腈及其水解的中间产物烟酰胺的水解反应动力学数据.结果表明,在不同的实验条件下,烟腈水解可以高选择性地得到烟酰胺或烟酸;烟腈水解为一级连串反应;烟腈和烟酰胺水解反应活化能分别为652和858 kJ/mol;本研究为烟酸和烟酰胺的绿色制备以及高温液态水中腈类化合物水解规律的探讨提供了重要的基础数据.     

腺嘌呤类细胞分裂素的合成及结构-活性关系

讨论了腺嘌呤类细胞分裂素的合成方法及其结构-活性关系。     

Flavin adenine dinucleotide binding is the crucial step in alcohol oxidase assembly in the yeast Hansenula polymorpha

We have studied the role of flavin adenine dinucleotide (FAD) in the in vivo assembly of peroxisomal alcohol oxidase (AO) in the yeast Hansenula polymorpha. In previous studies, using a riboflavin (Rf) autotrophic mutant, an unequivocal judgement could not be made, since Rf-limitation led to a partial block of AO import in this mutant. This resulted in the accumulation of AO precursors in the cytosol where they remained separated from the putative peroxisomal AO assembly factors. In order to circumvent the peroxisomal membrane barrier, we have now studied AO assembly in a peroxisome-deficient/Rf-autotrophic double mutant (Δper1.rif1) of H. polymorpha. By sucrose density centrifugation and native gel electrophoresis, three conformations of AO were detected in crude extracts of Δper1.rif1 cells grown under Rf-limitation, namely active octameric AO and two inactive, monomeric forms. One of the latter forms lacked FAD; this form was barely detectable in extracts wild-type and Δper1 cells, but had accumulated in the cytosol of rif1 cells. The second form of monomeric AO contained FAD; this form was also present in Δper1 cells but absent/very low in wild-type and rif1 cells. In vivo only these FAD-containing monomers associate into the active, octameric protein. We conclude that in H. polymorpha FAD binding to the AO monomer is mediated by a yet unknown peroxisomal factor and represents the crucial and essential step to enable AO oligomerization; the actual octamerization and the eventual crystallization in peroxisomes most probably occurs spontaneously.     

卤素负载锌-腺嘌呤骨架材料的构建及无助剂催化CO2环加成反应

富氮型金属有机骨架材料（MOFs）具有良好的CO₂捕集性能,但其CO₂催化性能常需添加带氢键或亲核基团的助催化剂。以硝酸锌-腺嘌呤-异烟酸［Zn（NO₃）₂-Ad-Int-DMF］构建稳健的骨架材料,发现其催化合成碳酸丙烯酯（PC）收率不足2%;尝试选型锌盐前体引入亲核卤素,在DMF溶剂中部分卤代锌盐不能络合结晶成MOFs,而形成的ZnI₂-Ad-Int-DMF收率提升至19.5%;溶剂由DMF调变为H₂O-DMF混合溶剂,H₂O的引入避免了卤素对金属和配体间络合的影响,使得以ZnCl₂、ZnBr₂和ZnI₂为前体均能晶化成MOFs,其热分解起始温度（Tonset,434℃）明显高于ZnI₂-Ad-Int-DMF（280℃）,而比表面积（<14 m²/g）明显小于后者（571 m²/g）,造成活性低于后者。通过CO₂吸附脉冲和UV-Vis漫反射推测I^-由于与Zn碱位的相互作用而均匀吸附于骨架上。进而,在无助剂、无溶剂工况下对CO₂环加成反应进行活性评价。温度升高显著提升PC产率,140℃下ZnI₂-Ad-Int-DMF催化产率可达98.5%,但由于吸附在骨架上的卤素在高温、溶剂环境下会脱落,造成重复性实验中活性下降,而该类MOFs在反应前后骨架保持稳定,未造成坍塌或明显孔堵塞现象。后续如能强化卤素的吸附或温和化反应条件,则具有良好的应用潜力。     

CRIP1生物学功能的定量蛋白质组学分析

CRIP1（cysteine-rich intestinal protein 1）是含有双锌指结构域的蛋白,在很多肿瘤细胞中高表达,但其在肺癌细胞中的生理学功能尚不明确。本研究应用定量蛋白质组学探究CRIP1过表达对肺癌顺铂耐药细胞（A549/DDP）的影响及作用机制。运用慢病毒载体系统构建了CRIP1过表达的A549/DDP稳转细胞系,利用Western Blotting证实了单克隆细胞系中CRIP1的过表达。发现CRIP1过表达能够加快细胞增殖,增加细胞的耐药性。通过定量蛋白质组学分析,鉴定了CRIP1过表达引起的蛋白质组的变化,并且对上调和下调的蛋白进行聚类分析。结果表明,CRIP1过表达上调了烟酰胺磷酸核糖转移酶（NAMPT）和NAD依赖型氧化还原酶的表达,从而促进细胞的增殖,并且提高了细胞的耐药性。     

The Metabolism of Fatty Alcohols in Lipid Nanoparticles by Alcohol Dehydrogenase

ABSTRACT

Fatty alcohols are commonly used in lipid-based drug delivery systems including parenteral emulsions and solid lipid nanoparticles (NPs). The purpose of these studies was to determine whether horse liver alcohol dehydrogenase (HLADH), a NAD-dependent enzyme, could metabolize the fatty alcohols within the NPs and thus serve as a mechanism to degrade these NPs in the body. Solid nanoparticles (<100 nm) were engineered from oil-in-water microemulsion precursors using emulsifying wax NF as the oil phase and polyoxyethylene 20-stearyl ether (Brij 78) as the surfactant. Emulsifying wax contains both cetyl and stearyl alcohols. NPs were incubated with the enzyme and NAD⁺ at 37°C for up to 48 h, and the concentrations of fatty alcohols were quantitatively determined over time by gas chromatography (GC). The concentrations of cetyl alcohol and stearyl alcohol within the NPs decreased to only 10–20% remaining after 15–24 h of incubation. In parallel, NP size, turbidity and the fluorescence intensity of NADH all increased over time. It was concluded that horse liver alcohol dehydrogenase/NAD⁺ was able to metabolize the fatty alcohols within the NPs, suggesting that NPs made of fatty alcohols may be metabolized in the body via endogenous alcohol dehydrogenase enzyme systems.     

Thermodynamics of noncovalent interactions in hydrophobically‐substituted water‐soluble polymers from intrinsic viscosity measurements: Application to nucleobase‐substituted pullulans

Hydrophobically substituted water‐soluble polymers (HSWSP) act as associative thickeners through the reversible crosslinking from noncovalent interactions between the various groups on the polymer chains in aqueous solution. This article shows how the intrinsic viscosity (IV) of nonionic HSWSP can be used to define the thermodynamics of these interactions. Literature data on the IV of pullulans substituted by nucleobase ester groups (thyminylbutyryl and adeninylbutyryl) (Mocanu et al., Can J Chem, 1995, 73, 1933) are used as an exemplar of these procedures. The intramolecular crosslinking in these substituted pullulans is deduced to be “unimolecular” (association constant K₁ = 1 M^?1), as contrasted with the “bimolecular” behavior expected from the stacking of the free nucleobases; evidently the crosslinking results from hydrophobic interactions between the butyryl linking groups and the main chain. The results are compared with those from other HSWSP, and from cosolute binding systems. The use of the water–octanol partition coefficients of model systems to elucidate hydrophobic interactions in HSWSP, and of denaturant cosolutes (especially urea) to diagnose the presence and strength of these interactions, are also discussed. Emphasis is placed on the need for further such studies to identify the interactions underlying the rheological behavior of the nonionic HSWSP, and of the more common ionic types. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011