Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.     

New method for peptide purification based on selective removal of truncation peptide impurities after SPPS with orthogonal capping

Peptide purification by high-performance liquid chromatography (HPLC) is associated with high solvent consumption, relatively large effort and lack of efficient parallelization. As an alternative, many catch-and-release (c&r) purification methods have been developed over the last decades to enable the efficient parallel purification of peptides originating from solid-phase peptide synthesis (SPPS). However, with one exception, none of the c&r systems has been widely established in industry and academia until today. Herein, we present an entirely new chromatography-free purification concept for peptides synthesized on a solid support, termed reactive capping purification (RCP). The RCP method relies on the capping of truncation peptides arising from incomplete coupling of amino acids during SPPS with a reactive tag. The reactive tag contains a masked functionality that, upon liberation during cleavage from the resin, enables straightforward purification of the peptide by incubation with a resin-bound reactive moiety. In this work, two different reactive tags based on masked thiols were developed. Capping with these reactive tags during SPPS led to effective modification of truncated sequences and subsequent removal of the latter by chemoselective reaction with a maleimide-functionalized solid support. By introducing a suitable protecting group strategy, the thiol-based RCP method described here could also be successfully applied to a thiol-containing peptide. Finally, the purification of a 15-meric peptide by the RCP method was demonstrated. The developed method has low solvent consumption, has the potential for efficient parallelization, uses readily available reagents, and is experimentally simple to perform.     

Plants as sources of natural and recombinant anti-cancer agents

Herbal remedies were the first medicines used by humans due to the many pharmacologically active secondary metabolites produced by plants. Some of these metabolites inhibit cell division and can therefore be used for the treatment of cancer, e.g. the mitostatic drug paclitaxel (Taxol). The ability of plants to produce medicines targeting cancer has expanded due to the advent of genetic engineering, particularly in recent years because of the development of gene editing systems such as the CRISPR/Cas9 platform. These technologies allow the introduction of genetic modifications that facilitate the accumulation of native pharmaceutically-active substances, and even the production heterologous recombinant proteins, including human antibodies, lectins and vaccine candidates. Here we discuss the anti-cancer agents that are produced by plants naturally or following genetic modification, and the potential of these products to supply modern healthcare systems. Special emphasis will be put on proteinaceous anti-cancer agents, which can exhibit an improved selectivity and reduced side effects compared to small molecule-based drugs.     

聚乙二醇对平邑甜茶叶片气孔器超微结构的影响

 采用透射电镜技术对聚乙二醇（PEG）模拟干旱处理的平邑甜茶(Malus hupehensis Rehd (pingyitiancha))叶片气孔器超微结构进行了观察,并对叶片气孔器超微结构制片方法进行了改进。所观察结果如下：1）PEG胁迫后保卫细胞中叶绿体数量增加,而叶绿体中淀粉粒数量呈下降趋势;2）PEG胁迫降低保卫细胞中线粒体的数量及破坏线粒体的超微结构;3）PEG胁迫使平邑甜茶保卫细胞中的液泡变小以致不明显。     

Transgenic Japanese lawngrass (Zoysia japonica Steud.) plants regenerated from protoplasts

Transgenic Japanese lawngrass (Zoysia japonica Steud.) plants were generated by means of polyethylene glycol (PEG)-mediated direct gene transfer into protoplasts. The plasmid pBC1 was used to deliver the hygromycin phosphotransferase (hph) and β-glucuronidase (gus) genes into protoplasts. Selection with a high concentration (400 mg/l) of hygromycin yielded a number of resistant calli and about 400 plants were generated. Polymerase chain reaction (PCR) and Southern hybridization analyses revealed that all of then plants tested contained introduced genes. The gus gene regulated by the maize alcohol dehydrogenase-1 (Adh 1) promoter was expressed in the leaves and roots of transgenic Japanese lawngrass plants. Received: 13 December 1996 / Revision received: 9 June 1997 / Accepted: 2 September 1997     

A stable Escherichia coli-mycobacteria shuttle vector 'pSO246' in Mycobacterium bovis BCG

Abstract The most widely used plasmid vector system in mycobacteria is based on pAL5000 from Mycobacterium fortuitum . The derivatives of the pAL5000-based shuttle vectors between Escherichia coli and mycobacteria, which we have utilized to secrete recombinant antigens, were generated. The stability of the vectors was assessed in Mycobacterium bovis BCG (BCG). The plasmid vector pSO246 was stable in BCG for at least 50 generations.     

Structural Basis for the SUMO2 Isoform Specificity of SENP7

SUMO proteases or deSUMOylases regulate the lifetime of SUMO-conjugated targets in the cell by cleaving off the isopetidic bond between the substrate and the SUMO modifier, thus reversing the conjugation activity of the SUMO E3 ligases. In humans the deSUMOylating activity is mainly conducted by the SENP/ULP protease family, which is constituted of six members sharing a homologous catalytic globular domain. SENP6 and SENP7 are the most divergent members of the family and they show a unique SUMO2/3 isoform preference and a particular activity for dismantling polySUMO2 chains. Here, we present the crystal structure of the catalytic domain of human SENP7 bound to SUMO2, revealing structural key elements for the SUMO2 isoform specificity of SENP7. In particular, we describe the specific contacts between SUMO2 and a unique insertion in SENP7 (named Loop1) that is responsible for the SUMO2 isoform specificity. All the other interface contacts between SENP7 and SUMO2, including the SUMO2 C-terminal tail interaction, are conserved among members of the SENP/ULP family. Our data give insight into an evolutionary adaptation to restrict the deSUMOylating activity in SENP6 and SENP7 for the SUMO2/3 isoforms.     

高浓度玻璃酸钠滴眼液与聚乙二醇滴眼液防治飞秒激光辅助LASIK术后干眼的临床疗效对比

目的:比较高浓度玻璃酸钠滴眼液与聚乙二醇滴眼液防治飞秒激光辅助LASIK术后干眼的临床效果。方法:选取2016年1月至2017年1月在我院视光学中心收治的飞秒激光辅助LASIK术后干眼患者80例并将其随机分为A、B两组,分别给予玻璃酸钠滴眼液(3 g/L)、聚乙二醇滴眼液,在用药后1周、2周、1个月进行干眼体征检查,比较患者用药前后泪液分泌试验(SIT)、泪膜破裂时间(BUT)、角膜荧光素染色(FL)的变化。结果:术后1周,所有患者BUT均较术前显著降低,FL均较术前显著升高,差异均有统计学意义(P0.05),但SIT与术前比较差异无统计学意义(P0.05)。两组患者用药后SIT数值随时间变化差异没有统计学意义(P0.05);A组和B组分别在用药后1周、2周、1个月时进行比较,SIT变化差异没有统计学意义(P0.05);而A组BUT时间、FL评分改善时间明显早于B组,差异有统计学意义(P0.05)。结论:滴用高浓度玻璃酸钠滴眼液(3 g/L)对飞秒激光辅助LASIK术后干眼患者的效果明显优于滴用聚乙二醇。     

Structural and biochemical characterization of a novel aldehyde dehydrogenase encoded by the benzoate oxidation pathway in Burkholderia xenovorans LB400

The recently identified benzoate oxidation (box) pathway in Burkholderia xenovorans LB400 (LB400 hereinafter) assimilates benzoate through a unique mechanism where each intermediate is processed as a coenzyme A (CoA) thioester. A key step in this process is the conversion of 3,4-dehydroadipyl-CoA semialdehyde into its corresponding CoA acid by a novel aldehyde dehydrogenase (ALDH) (EC 1.2.1.x). The goal of this study is to characterize the biochemical and structural properties of the chromosomally encoded form of this new class of ALDHs from LB400 (ALDH_C) in order to better understand its role in benzoate degradation. To this end, we carried out kinetic studies with six structurally diverse aldehydes and nicotinamide adenine dinucleotide (phosphate) (NAD ⁺ and NADP ⁺). Our data definitively show that ALDH_C is more active in the presence of NADP ⁺ and selective for linear medium-chain to long-chain aldehydes. To elucidate the structural basis for these biochemical observations, we solved the 1.6-Å crystal structure of ALDH_C in complex with NADPH bound in the cofactor-binding pocket and an ordered fragment of a polyethylene glycol molecule bound in the substrate tunnel. These data show that cofactor selectivity is governed by a complex network of hydrogen bonds between the oxygen atoms of the 2′-phosphoryl moiety of NADP ⁺ and a threonine/lysine pair on ALDH_C. The catalytic preference of ALDH_C for linear longer-chain substrates is mediated by a deep narrow configuration of the substrate tunnel. Comparative analysis reveals that reorientation of an extended loop (Asn478-Pro490) in ALDH_C induces the constricted structure of the substrate tunnel, with the side chain of Asn478 imposing steric restrictions on branched-chain and aromatic aldehydes. Furthermore, a key glycine (Gly104) positioned at the mouth of the tunnel allows for maximum tunnel depth required to bind medium-chain to long-chain aldehydes. This study provides the first integrated biochemical and structural characterization of a box-pathway-encoded ALDH from any organism and offers insight into the catalytic role of ALDH_C in benzoate degradation.