A facile method for controlling the reaction equilibrium of sphingolipid ceramide N-deacylase for lyso-glycosphingolipid production

Lyso-glycosphingolipids (lyso-GSLs), the N-deacylated forms of glycosphingolipids (GSLs), are important synthetic intermediates for the preparation of GSL analogs. Although lyso-GSLs can be produced by hydrolyzing natural GSLs using sphingolipid ceramide N-deacylase (SCDase), the yield for this reaction is usually low because SCDase also catalyzes the reverse reaction, ultimately establishing an equilibrium between hydrolysis and synthesis. In the present study, we developed an efficient method for controlling the reaction equilibrium by introducing divalent metal cation and detergent in the enzymatic reaction system. In the presence of both Ca²⁺ and taurodeoxycholate hydrate, the generated fatty acids were precipitated by the formation of insoluble stearate salts and pushing the reaction equilibrium toward hydrolysis. The yield of GM1 hydrolysis can be achieved as high as 96%, with an improvement up to 45% compared with the nonoptimized condition. In preparative scale, 75 mg of lyso-GM1 was obtained from 100 mg of GM1 with a 90% yield, which is the highest reported yield to date. The method can also be used for the efficient hydrolysis of a variety of GSLs and sphingomyelin. Thus, this method should serve as a facile, easily scalable, and general tool for lyso-GSL production to facilitate further GSL research.     

Narrowband Light Total Antireflection and Absorption in Metal Film–Array Structures by Plasmonic Near-Field Coupling

We study the cooperative effects between plasmon gap modes and optical cavity modes of a novel triple-layer structure consisting of double continuous gold films separated by a gold nanosphere array. Narrowband near-perfect antireflection of optical field is achieved for the first time due to the strong near-field light–matter interaction within the deep sub-wavelength gaps between adjacent nanospheres combined with the spatial field confinement effects of the optical cavity built by the double gold films. The coexistence cooperation of near-field dipole plasmon resonances and spatial optical field confinement presents more efficient light modification than that of the individual subsystem and may open a new approach to manage light flow. By varying the period of nanosphere array, the diameter of nanospheres, and the distance between the array and the film, optical behaviors of the proposed structure can be tuned in a wide range. High environmental sensitivity and large figure of merit factor are obtained using this structure as the detecting substrate. Furthermore, ultra-compact structure and high conduction suggest the proposed structure being a good candidate for potential applications in highly integrated optoelectronic devices, such as plasmonic filters and sensors.     

A novel thermophilic endo-β-1,4-mannanase from Aspergillus nidulans XZ3: functional roles of carbohydrate-binding module and Thr/Ser-rich linker region

The gene man5XZ3 from Aspergillus nidulans XZ3 encodes a multimodular β-mannanase of glycoside hydrolase family 5 that consists of a family 1 carbohydrate-binding module (CBM1), a Thr/Ser-rich linker region, and a catalytic domain. Recombinant Man5XZ3 and its two truncated derivatives, Man5ΔCBM (removing the CBM1) and Man5ΔCL (removing both the CBM1 and linker region), were produced in Pichia pastoris and showed significant variance in the secondary structure. The three enzymes had similar biochemical properties, such as optimal pH and temperature (pH 5.0 and 80 °C) and excellent pH stability at pH 4.0–10.0. Removal of the CBM1 alone could improve the thermostability of Man5XZ3, but further removal of the linker region resulted in worse thermostability. Man5XZ3 retained greater enzyme activity in the presence of an organic solvent (acetone), two detergents (SDS and Triton X-100), and a chaotropic agent (urea) compared with Man5ΔCBM and Man5ΔCL. This study provides an excellent β-mannanase candidate favorable for various industries and primarily demonstrates the relationship between enzyme structure and function.     

Genome engineering of Agrobacterium tumefaciens using the lambda Red recombination system

Agrobacterium tumefaciens has been widely used as a tool for transgenesis in plants. The availability of its genome sequence should facilitate the directed engineering of improved properties; however, the current genome engineering options are laborious. Here, we investigated whether the lambda R ed operon can be applied for recombineering of the A. tumefaciens genome. First, we built an expression plasmid for A. tumefaciens employing a tetracycline-inducible promoter to regulate the Red operon. This multicopy plasmid was then itself modified in A. tumefaciens to verify that the Red operon was functional. Then, we modified the endogenous A. tumefaciens tumor-inducing plasmid and the linear chromosome. These results extend recombineering technology to a new host and indicate a fast and convenient way to engineer the A. tumefaciens genome for functional genomics and strain improvements.     

猕猴桃驯化改良百年启示及天然居群遗传渐渗的基因发掘

猕猴桃(Actinidia chinensis Planch.)是近代由野生到人工商品化栽培最为成功的植物驯化范例。该文综述了我国猕猴桃资源流失国外及其驯化改良和产业发展的100年历史, 概述了我国野生猕猴桃资源的分布状况, 并在总结我国猕猴桃品种选育改良、科研与产业发展的基础上, 阐明了我国猕猴桃资源及其发掘利用对世界猕猴桃产业的重要影响。通过深入分析野生猕猴桃资源的可持续发掘利用问题, 提出了利用天然杂交带的遗传渐渗作用开展野生植物新种质和新基因型发掘的思路。     

Exploring the stereochemistry of CXCR4-peptide recognition and inhibiting HIV-1 entry with D-peptides derived from chemokines

Chemokine receptor CXCR4 plays an important role in the immune system and the cellular entry of human immunodeficiency virus type 1 (HIV-1). To probe the stereospecificity of the CXCR4-ligand interface, d-amino acid peptides derived from natural chemokines, viral macrophage inflammatory protein II (vMIP-II) and stromal cell-derived factor-1alpha (SDF-1alpha), were synthesized and found to compete with (125)I-SDF-1alpha and monoclonal antibody 12G5 binding to CXCR4 with potency and selectivity comparable with or higher than their l-peptide counterparts. This was surprising because of the profoundly different side chain topologies between d- and l-enantiomers, which circular dichroism spectroscopy showed adopt mirror image conformations. Further direct binding experiments using d-peptide labeled with fluorescein (designated as FAM-DV1) demonstrated that d- and l-peptides shared similar or at least overlapping binding site(s) on the CXCR4 receptor. Structure-activity analyses of related peptide analogs of mixed chiralities or containing alanine replacements revealed specific residues at the N-terminal half of the peptides as key binding determinants. Acting as CXCR4 antagonists and with much higher biological stability than l-counterparts, the d-peptides showed significant activity in inhibiting the replication of CXCR4-dependent HIV-1 strains. These results show the remarkable stereochemical flexibility of the CXCR4-peptide interface. Further studies to understand the mechanism of this unusual feature of the CXCR4 binding surface might aid the development of novel CXCR4-binding molecules like the d-peptides that have high affinity and stability.     

Carbohydrate moiety of the Petunia inflata S3 protein is not required for self-incompatibility interactions between pollen and pistil.

For Petunia inflata and Nicotiana alata, which display gametophytic self-incompatibility, S proteins (the products of the multiallelic S gene in the pistil) have been shown to control the pistil's ability to recognize and reject self-pollen. The biochemical mechanism for rejection of self-pollen by S proteins has been shown to involve their ribonuclease activity; however, the molecular basis for self/non-self recognition by S proteins is not yet understood. Here, we addressed whether the glycan chain of the S3 protein of P. inflata is involved in self/non-self recognition by producing a nonglycosylated S3 protein in transgenic plants and examining the effect of deglycosylation on the ability of the S3 protein to reject S3 pollen. The S3 gene was mutagenized by replacing the codon for Asn-29, which is the only potential N-glycosylation site of the S3 protein, with a codon for Asp, and the mutant S3 gene was introduced into P. inflata plants of the S1S2 genotype. Six transgenic plants that produced a normal level of the nonglycosylated S3 protein acquired the ability to reject S3 pollen completely. These results suggest that the carbohydrate moiety of the S3 protein does not play a role in recognition or rejection of self-pollen and that the S allele specificity determinant of the S3 protein and those S proteins that contain a single glycan chain at the same site as the S3 protein must reside in the amino acid sequence itself.