Dimeric Crystal Structure of Rabbit l-Gulonate 3-Dehydrogenase/λ-Crystallin: Insights into the Catalytic Mechanism

l-Gulonate 3-dehydrogenase (GDH) is a bifunctional dimeric protein that functions not only as an NAD⁺-dependent enzyme in the uronate cycle but also as a taxon-specific λ-crystallin in rabbit lens. Here we report the first crystal structure of GDH in both apo form and NADH-bound holo form. The GDH protomer consists of two structural domains: the N-terminal domain with a Rossmann fold and the C-terminal domain with a novel helical fold. In the N-terminal domain of the NADH-bound structure, we identified 11 coenzyme-binding residues and found 2 distinct side-chain conformers of Ser124, which is a putative coenzyme/substrate-binding residue. A structural comparison between apo form and holo form and a mutagenesis study with E97Q mutant suggest an induced-fit mechanism upon coenzyme binding; coenzyme binding induces a conformational change in the coenzyme-binding residues Glu97 and Ser124 to switch their activation state from resting to active, which is required for the subsequent substrate recruitment. Subunit dimerization is mediated by numerous intersubunit interactions, including 22 hydrogen bonds and 104 residue pairs of van der Waals interactions, of which those between two cognate C-terminal domains are predominant. From a structure/sequence comparison within GDH homologues, a much greater degree of interprotomer interactions (both polar and hydrophobic) in the rabbit GDH would contribute to its higher thermostability, which may be relevant to the other function of this enzyme as λ-crystallin, a constitutive structural protein in rabbit lens. The present crystal structures and amino acid mutagenesis studies assigned the role of active-site residues: catalytic base for His145 and substrate binding for Ser124, Cys125, Asn196, and Arg231. Notably, Arg231 participates in substrate binding from the other subunit of the GDH dimer, indicating the functional significance of the dimeric state. Proper orientation of the substrate-binding residues for catalysis is likely to be maintained by an interprotomer hydrogen-bonding network of residues Asn196, Gln199, and Arg231, suggesting a network-based substrate recognition of GDH.     

European post-glacial forests: compositional changes in response to climatic change

Abstract. Multivariate analysis of an extensive palyno-logical database for Europe has enabled reconstruction of broad-scale vegetation history. Whereas many major features of present vegetation patterns were established early in the Holocene, floristic composition of the forests has changed continuously up to the present day. For example, although ‘mixed deciduous forests’ had reached approximately their present extent in northwest Europe by 8000 B.P., Tilia peaked in abundance in these forests during the middle post glacial, whereas Pinus was most abundant in these forests during the early post-glacial and Fagus increased in abundance only in recent millennia. Pollen-climate response surfaces for major pollen taxa show how their distribution and abundance patterns relate to contemporary climate. Past forest-compositional changes were responses to climatic changes, the nature of which can be inferred from pollen-climate response surfaces. Post-glacial climate changes have been different in magnitude and direction in different regions of Europe. For example, in recent millennia the vegetation changes indicate decreasing summer temperatures in northern Europe but increasing summer temperatures in the Mediterranean region. The way in which vegetation responded to past climatic changes gives insight into the likely response of vegetation to future climate changes induced by the ‘greenhouse effect’.     

Protein A-based antibody immobilization onto polymeric microdevices for enhanced sensitivity of enzyme-linked immunosorbent assay

Highly efficient antibody immobilization is extremely crucial for the development of high-performance polymeric microdevices for enzyme-linked immunosorbent assay (ELISA). In this article, a site-selective tyrosinase (TR)-catalyzed protein A strategy for antibody immobilization was developed to enhance the sensitivity of ELISA in poly-(methyl methacrylate) (PMMA) microchannels for interferon-gamma (IFN-gamma) assay. To effectively immobilize the target antibodies, oxygen plasma was first used to activate the inert PMMA. This is followed by poly(ethyleneimine) (PEI) coating, an amine-containing functional polymer. For comparison, protein A was also immobilized through the commonly used amine-glutaraldehyde (GA) chemistry. Oxygen plasma treatment effectively increased the amount of PEI attachment and subsequent binding efficiency of the primary antibody. The antibody immobilized via TR-catalyzed protein A was able to provide much better specific antigen capture efficiency than GA chemistry due to the optimal spacing and orientation. Consequently, by using this new method, the detection signal and the signal-to-noise ratio of the ELISA immunoassay in microdevices were all significantly improved. In comparison to the standard assay carried out in the 96-well microtiter plate, the treated microchannels exhibited a broader detection range and a shorter detection time. And the detection limit was also decreased to 20 pg/mL, much lower than that obtained in other microdevices.     

深共熔溶剂提取杨树桑黄多酚类物质工艺优化及提取物抗氧化活性研究

桑黄是一类应用广泛的药用真菌,桑黄多酚具有抗氧化、抗炎和降糖作用。本研究中的杨树桑黄属于桑黄中重要且能栽培的一种。本研究采用深共熔溶剂(deep eutectic solvent,DES)从栽培杨树桑黄子实体中提取多酚类化合物,考察了不同提取条件对提取率的影响。采用氯化胆碱与尿素组成的DES体系对多酚进行提取,并进一步采用响应面法对提取条件进行优化,获得最佳提取工艺参数为21 min、80 ℃、料液比1:260 g/mL。在此条件下,多酚的提取率高达(23.17±0.88) mg/g,远高于传统方法(12.45±1.88) mg/g。最优条件提取的多酚显示了很强的DPPH和ABTS的清除能力。由此可见,采用DES法从杨树桑黄子实体中提取酚类化合物比传统方法更有效。     

樟叶越桔细胞悬浮培养条件的优化

为了提高樟叶越桔(Vacciniumdunalianum)悬浮培养细胞的生物量,以樟叶越桔叶片愈伤组织为试材,通过单因素试验探究不同蔗糖浓度、培养基pH值、培养基体积、初始接种量和摇床转速对悬浮培养细胞生长的影响,并根据响应面法Box-Behnken试验设计原理进行组合试验以优化培养条件。结果显示,以改良WPM培养基为基础培养基,樟叶越桔细胞悬浮培养的最优条件为40 g·L^–1蔗糖、培养基pH5.2、培养基体积45 mL、初始接种量2.64 g和摇床转速为149 r·min^–1,其细胞生物量干重为0.184 4 g,与理论预测值0.184 5 g较为接近,且细胞的生长曲线呈S型。研究结果为樟叶越桔悬浮培养细胞次生代谢产物的生产调控奠定了技术基础。     

Angiogenic factor AGGF1 blocks neointimal formation after vascular injury via interaction with integrin α7 on vascular smooth muscle cells

Angiogenic factor AGGF1 (AngioGenic factor with G-patch and FHA (Forkhead-Associated) domain 1) blocks neointimal formation (formation of a new or thickened layer of arterial intima) after vascular injury by regulating phenotypic switching of vascular smooth muscle cells (VSMCs). However, the AGGF1 receptor on VSMCs and the underlying molecular mechanisms of its action are unknown. In this study, we used functional analysis of serial AGGF1 deletions to reveal the critical AGGF1 domain involved in VSMC phenotypic switching. This domain was required for VSMC phenotypic switching, proliferation, cell cycle regulation, and migration, as well as the regulation of cell cycle inhibitors cyclin D, p27, and p21. This domain also contains an RDDAPAS motif via which AGGF1 interacts with integrin α7 (ITGA7), but not α8. In addition, we show that AGGF1 enhanced the expression of contractile markers MYH11, α-SMA, and SM22 and inhibited MEK1/2, ERK1/2, and ELK phosphorylation in VSMCs, and that these effects were inhibited by knockdown of ITGA7, but not by knockdown of ITGA8. In vivo, deletion of the VSMC phenotypic switching domain in mice with vascular injury inhibited the functions of AGGF1 in upregulating α-SMA and SM22, inhibiting MEK1/2, ERK1/2, and ELK phosphorylation, in VSMC proliferation, and in blocking neointimal formation. Finally, we show the inhibitory effect of AGGF1 on neointimal formation was blocked by lentivirus-delivered shRNA targeting ITGA7. Our data demonstrate that AGGF1 interacts with its receptor integrin α7 on VSMCs, and this interaction is required for AGGF1 signaling in VSMCs and for attenuation of neointimal formation after vascular injury.     

SDS-capped 1-pyrenecarboxaldehyde nanoprobe for selective detection of Cu2+ ion from water samples: Spectroscopic approach

Sodium dodecyl sulfate (SDS)-capped 1-pyrenecarboxaldehyde nanoparticles (PyalNPs) were prepared using a reprecipitation method in an aqueous medium and exhibited red-shifted aggregation-induced enhanced emission (AIEE). The dynamic light scattering (DLS) examination showed narrower particle size distribution with an average particle size of 41 nm, whereas −34.5 mV zeta potential value indicate the negative surface charge and good stability of nanoparticles (NPs) in an aqueous medium. The AIEE was seen at λ_max = 473 nm in a fluorescence spectrum of a PyalNP suspension. In the presence of Cu²⁺ ions, the fluorescence of PyalNPs quenches very significantly, even in the presence of other metal ions like Ba²⁺, Ca²⁺, Cd²⁺, Co²⁺, Al³⁺, Fe²⁺, Hg²⁺, Ni²⁺ and Mg²⁺. The changes in the fluorescence lifetime of PyalNPs in the presence of Cu²⁺ ions suggested that the type of quenching was dynamic. The fluorescence quenching data for the NPs suspension fitted well into a typical Stern–Volmer relationship in the concentration range 1.0–25 μg/ml of Cu²⁺ ions. The estimated value of the correlation coefficient R² = 0.9877 was close to 1 and showed the linear relationship between quenching data and Cu²⁺ ion concentration. The limit of detection (LOD) was found to be 0.94 ng/ml and is far below the tolerable intake limit value of 1.3 μg/ml accepted by the World Health Organization for Cu²⁺ ions in drinking water. The fluorescence quenching approach for a SDS-capped Pyal nanosuspension for copper ion quantification is of high specificity and coexisting ions were found to interfere very negligibly. The developed method was successfully applied for the estimation of copper ions in river water samples.     

Cooling Induced Surface Reconstruction during Synthesis of High‐Ni Layered Oxides

Transition metal layered oxides have been the dominant cathodes in lithium‐ion batteries, and among them, high‐Ni ones (LiNi_xMn_yCo_zO₂; x ≥ 0.7) with greatly boosted capacity and reduced cost are of particular interest for large‐scale applications. The high Ni loading, on the other hand, raises the critical issues of surface instability and poor rate performance. The rational design of synthesis leading to layered LiNi_0.7Mn_0.15Co_0.15O₂ with greatly enhanced rate capability is demonstrated, by implementing a quenching process alternative to the general slow cooling. In situ synchrotron X‐ray diffraction, coupled with surface analysis, is applied to studies of the synthesis process, revealing cooling‐induced surface reconstruction involving Li₂CO₃ accumulation, formation of a Li‐deficient layer and Ni reduction at the particle surface. The reconstruction process occurs predominantly at high temperatures (above 350 °C) and is highly cooling‐rate dependent, implying that surface reconstruction can be suppressed through synthetic control, i.e., quenching to improve the surface stability and rate performance of the synthesized materials. These findings may provide guidance to rational synthesis of high‐Ni cathode materials.