Functional Expression in Escherichia coli of the Disulfide-Rich Sea Anemone Peptide APETx2, a Potent Blocker of Acid-Sensing Ion Channel 3

Acid-sensing ion channels (ASICs) are proton-gated sodium channels present in the central and peripheral nervous system of chordates. ASIC3 is highly expressed in sensory neurons and plays an important role in inflammatory and ischemic pain. Thus, specific inhibitors of ASIC3 have the potential to be developed as novel analgesics. APETx2, isolated from the sea anemone Anthopleura elegantissima, is the most potent and selective inhibitor of ASIC3-containing channels. However, the mechanism of action of APETx2 and the molecular basis for its interaction with ASIC3 is not known. In order to assist in characterizing the ASIC3-APETx2 interaction, we developed an efficient and cost-effective Escherichia coli periplasmic expression system for the production of APETx2. NMR studies on uniformly (13)C/(15)N-labelled APETx2 produced in E. coli showed that the recombinant peptide adopts the native conformation. Recombinant APETx2 is equipotent with synthetic APETx2 at inhibiting ASIC3 channels expressed in Xenopus oocytes. Using this system we mutated Phe15 to Ala, which caused a profound loss of APETx2's activity on ASIC3. These findings suggest that this expression system can be used to produce mutant versions of APETx2 in order to facilitate structure-activity relationship studies.     

A Tale of Toxin Promiscuity: The Versatile Pharmacological Effects of Hcr 1b-2 Sea Anemone Peptide on Voltage-Gated Ion Channels

Sea anemones are a rich source of biologically active compounds. Among approximately 1100 species described so far, Heteractis crispa species, also known as sebae anemone, is native to the Indo-Pacific area. As part of its venom components, the Hcr 1b-2 peptide was first described as an ASIC1a and ASIC3 inhibitor. Using Xenopus laevis oocytes and the two-electrode voltage-clamp technique, in the present work we describe the remarkable lack of selectivity of this toxin. Besides the acid-sensing ion channels previously described, we identified 26 new targets of this peptide, comprising 14 voltage-gated potassium channels, 9 voltage-gated sodium channels, and 3 voltage-gated calcium channels. Among them, Hcr 1b-2 is the first sea anemone peptide described to interact with isoforms from the Kv7 family and T-type Cav channels. Taken together, the diversity of Hcr 1b-2 targets turns this toxin into an interesting tool to study different types of ion channels, as well as a prototype to develop new and more specific ion channel ligands.     

外源Ca2+及钙离子信号抑制剂对茶树抗寒性的影响

钙离子信号通路在植物抗寒响应中起着重要作用。为了解钙离子信号通路与茶树抗寒性之间的关系,采用人工气候室模拟低温胁迫和外源施用钙调素（CaM）抑制剂W-7[N-(6-Aminohexyl)-5-chloro-1 -naphthalenesulfonamide]、钙信号通道抑制剂LaCl₃及CaCl₂溶液的方法,测定了低温胁迫下茶树叶片各种与抗寒相关的生理指标的变化情况。检测结果表明,钙离子信号抑制剂W-7、LaCl₃处理均能提高茶树叶片相对电导率,提高茶树叶片中丙二醛（MDA）、超氧阴离子自由基（O₂·^-）、脯氨酸的含量,抑制超氧化物歧化酶（SOD）的活性;而CaCl₂溶液处理茶树叶片相对电导率及叶片中MDA、超氧阴离子自由基和脯氨酸的含量降低,SOD活性提高。表明钙离子信号系统在茶树抗寒过程中发挥了重要作用。     

Strategies for the Development of Conotoxins as New Therapeutic Leads

Peptide toxins typically bind to their target ion channels or receptors with high potency and selectivity, making them attractive leads for therapeutic development. In some cases the native peptide as it is found in the venom from which it originates can be used directly, but in many instances it is desirable to truncate and/or stabilize the peptide to improve its therapeutic properties. A complementary strategy is to display the key residues that make up the pharmacophore of the peptide toxin on a non-peptidic scaffold, thereby creating a peptidomimetic. This review exemplifies these approaches with peptide toxins from marine organisms, with a particular focus on conotoxins.     

Development of Highly Selective Kv1.3-Blocking Peptides Based on the Sea Anemone Peptide ShK

ShK, from the sea anemone Stichodactyla helianthus, is a 35-residue disulfide-rich peptide that blocks the voltage-gated potassium channel Kv1.3 at ca. 10 pM and the related channel Kv1.1 at ca. 16 pM. We developed an analog of this peptide, ShK-186, which is currently in Phase 1b-2a clinical trials for the treatment of autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. While ShK-186 displays a >100-fold improvement in selectivity for Kv1.3 over Kv1.1 compared with ShK, there is considerable interest in developing peptides with an even greater selectivity ratio. In this report, we describe several variants of ShK that incorporate p-phophono-phenylalanine at the N-terminus coupled with internal substitutions at Gln16 and Met21. In addition, we also explored the combinatorial effects of these internal substitutions with an alanine extension at the C-terminus. Their selectivity was determined by patch-clamp electrophysiology on Kv1.3 and Kv1.1 channels stably expressed in mouse fibroblasts. The peptides with an alanine extension blocked Kv1.3 at low pM concentrations and exhibited up to 2250-fold selectivity for Kv1.3 over Kv1.1. Analogs that incorporates p-phosphono-phenylalanine at the N-terminus blocked Kv1.3 with IC₅₀s in the low pM range and did not affect Kv1.1 at concentrations up to 100 nM, displaying a selectivity enhancement of >10,000-fold for Kv1.3 over Kv1.1. Other potentially important Kv channels such as Kv1.4 and Kv1.6 were only partially blocked at 100 nM concentrations of each of the ShK analogs.     

2011-2012年新疆维吾尔自治区亚麻品种试验总结

为适应新疆亚麻生产发展的需要，有必要对我区自育和引进的优质亚麻品种，在不同生态条件、不同种植区域的适应性、抗逆性和丰产性进行鉴定。伊犁亚麻试验站在具有代表性的伊宁市、昭苏县、新源县和尼勒克县4个传统亚麻种植区进行多年多点区域试验。由2011及2012年的试验结果可看出：品系2011-4和N-22在两年四个点的综合评价较好，表现较突出。     

Enhanced resistance to bacterial infection byErwinia carotovora subsp.atroseptica in transgenic potato plants expressing the attacin or the cecropin SB-37 genes

Blackleg and soft rot diseases, caused by the bacteriumErwinia carotovora, are among the diseases that cause important losses in culture and storage of potato. In this paper, we introduced bacterial resistance into potato, via genes encoding for proteins with antibacterial activity. For this purpose, potato clones were transformed either with the gene encoding the acidic attacin protein fromHyalophora cecropia, or with the gene encoding the cecropin analog peptide SB37. These clones were evaluated for soft rot and blackleg resistance, after inoculation with the bacterial strainErwinia carotovora subsp.atroseptica T7. Results reported in this paper indicate that a considerable percentage of the potato clones (15–22%) showed increased resistance to bacterial infection, revealed by reduced severity of blackleg or soft rot symptoms. Expression of the transgenes was demonstrated in some of the clones by Northern blot analysis. This is the first report indicating that expression of the gene encoding for an attacin protein and for the cecropin SB-37 peptide in transgenic potato confers increased resistance to bacterial infection.     

小麦细胞色素P450(TaP450)基因的克隆与序列分析

细胞色素P450是一种多功能氧化酶,在植物体内担当着生物合成、代谢解毒以及抗逆等重要功能。本研究采用RACE方法从普通小麦中同源克隆到一个新的P450基因,命名为TaP450(Genbank No.KJ541960)。该基因基因组序列全长为2 643bp,含有2个外显子和1个内含子,cDNA全长为2 033bp,含有一个1 557bp的开放读码框,推导蛋白含518个氨基酸;序列分析发现其具有保守的P450结构域,但该蛋白与已报道的小麦P450蛋白序列有较大差异,表明它是小麦P450家族的新成员;蛋白结构特征分析发现,该蛋白的分子量为57.092kD,等电点为8.63,N端具有一个含29个氨基酸的跨膜结构和一个含22个氨基酸的信号肽,亚细胞定位分析表明该蛋白属于分泌蛋白。其在小麦叶片、茎、根与种子中均有表达,但在叶片和茎中表达量最高;在胁迫处理下,该基因的表达量均上调,且在盐胁迫处理下上调尤为显著,表明该基因与盐胁迫密切相关。     

Mechanism of Action and Therapeutic Potential of the β-Hairpin Antimicrobial Peptide Capitellacin from the Marine Polychaeta Capitella teleta

Among the most potent and proteolytically resistant antimicrobial peptides (AMPs) of animal origin are molecules forming a β-hairpin structure stabilized by disulfide bonds. In this study, we investigated the mechanism of action and therapeutic potential of the β-hairpin AMP from the marine polychaeta Capitella teleta, named capitellacin. The peptide exhibits a low cytotoxicity toward mammalian cells and a pronounced activity against a wide range of bacterial pathogens including multi-resistant bacteria, but the mechanism of its antibacterial action is still obscure. In view of this, we obtained analogs of capitellacin and tachyplesin-inspired chimeric variants to identify amino acid residues important for biological activities. A low hydrophobicity of the β-turn region in capitellacin determines its modest membranotropic activity and slow membrane permeabilization. Electrochemical measurements in planar lipid bilayers mimicking the E. coli membrane were consistent with the detergent-like mechanism of action rather than with binding to a specific molecular target in the cell. The peptide did not induce bacterial resistance after a 21-day selection experiment, which also pointed at a membranotropic mechanism of action. We also found that capitellacin can both prevent E. coli biofilm formation and destroy preformed mature biofilms. The marked antibacterial and antibiofilm activity of capitellacin along with its moderate adverse effects on mammalian cells make this peptide a promising scaffold for the development of drugs for the treatment of chronic E. coli infections, in particular those caused by the formation of biofilms.     

Total synthesis of a marine alkaloid--rigidin E

In the present paper, we report an efficient total synthesis of a marine alkaloid, rigidin E. The key tetrasubstituted 2-amino-3-carboxamidepyrrole intermediate was synthesized by cascade Michael addition/intramolecular cyclization between N-(2-(4-(benzyloxy)phenyl)-2-oxoethyl)methanesulfonamide and 3-(4-(benzyloxy)phenyl)-2-cyano-N-methylacrylamide. Subsequent carbonylation with triphosgene catalyzed by I(2) and deprotection of benzyl groups afforded rigidin E in 21% overall yield. This strategy has the merits of metal-free reactions, low cost, mild reaction protocols, and easy access to diversity-oriented derivatives for potential structure-activity relationship investigation.     

木薯转化酶抑制子MeINH3基因的克隆及生物信息学分析

转化酶抑制子调控转化酶的活性,在植物的糖代谢过程中具有重要作用。为了研究木薯的转化酶抑制子,本实验利用木薯基因组数据库分析及RT-PCR方法,从木薯中分离了1个木薯转化酶抑制子MeINH3的cDNA序列。MeINH3序列长度为564 bp,包含528 bp的完整开放阅读框,编码127个氨基酸,N端有16个氨基酸残基的信号肽,4个保守的半胱氨酸残基可形成两个二硫桥。亚细胞定位预测表明,MeINH3蛋白定位于胞外。根据生物信息学分析结果,推测其可能抑制木薯细胞壁转化酶活性。     

Antigenicity of Wheat Prolamins: Detailed Epitope Analysis using a Panel of Monoclonal Antibodies

Putative continuous epitopes, recognised by five panels of monoclonal antibodies (MAb) with differing specificities for gliadins and glutenin subunits, were identified using overlapping nonapeptides. These peptides corresponded to the entire sequence of an α/β-gliadin, a γ-gliadin, an ω-prolamin (homologous to ω-gliadin), a low molecular weight glutenin subunit (L M_rGS) and several high molecular weight glutenin subunits (HM_r GS). Antibodies that bound to γ- or ω-gliadins, L M_rGS or HM_r GS bound to the peptides at similar concentrations used normally in direct ELISA, but little binding to the peptides was seen for several antibodies that bound specifically to small groups of α/β-gliadins. Epitopes for these antibodies in α/β-gliadin may be discontinuous (i.e. derived from amino acid residues that are brought together by folding of the polypeptide chain or by juxtaposition of two polypeptide chains), since binding of these antibodies to gliadins was greatly decreased following the reduction of intra-molecular disulphide bonds. While some regions in particular subunits were immunodominant, such as the cysteine–cysteine containing peptide found in the central domain of many prolamins, a diversity of reaction patterns was found. Cross-reaction of antibody with peptides from other prolamin families was often due to binding to a peptide having significant sequence homology, but in some cases no homology was obvious. Some major trends were as follows. Antibodies which bound to most or all H M_rGS recognised the central repeat region, while those that were selective for one or two subunits bound to epitopes in the unique N- and/or C-terminal domains. A high proportion of the epitopes recognised by MAb to α-, β-, ω-gliadins and L M_rGS contained cysteine; these MAb may be useful in detecting covalent binding sites within or between subunits. Although a number of MAb bound a wide range of gliadins and GS, several of these recognised single (and differing) epitopes in the target proteins. However, comparatively few MAb recognised epitopes from either the N- or C-terminal regions of the target proteins. Several explanations are possible; either these regions are buried in the immunogen and not accessible for antibody production or alternatively the repeat sequences are immunodominant.     

Expression of Anti-Lipopolysaccharide Factor Isoform 3 in Chlamydomonas reinhardtii Showing High Antimicrobial Activity

Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene (ALFPm3), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii, which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 μg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii-derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 μM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon, providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.     

香蕉NBS-LRR抗病基因类似序列的克隆和分析

根据大多数抗病基因的核苷酸结合区(NBS)和富含亮氨酸重复序列(LRR)保守区的氨基酸序列设计PCR简并引物,利用快速而又可行的简并PCR技术获得NBS-LRR类抗病基因类似序列。笔者利用该技术从香蕉cDNA中获得了一个NBS-LRR类抗病基因类似序列,命名为B-NB(SAY739270)。结构域分析发现,B-NBS含有NBS蛋白所具有的P-Loop、Kinase-2、Kinase-3a和下游疏水GLPLAL结构域(HD,GLPLAL)4个保守基序。聚类分析发现,B-NBS与已报道的6个植物抗病基因的NBS基序的遗传距离在21.7之内。因此,B-NBS是一个香蕉NBS-LRR类抗病基因类似序列。本研究将为香蕉抗病基因克隆和抗病机理研究提供新的抗病候选基因资源。     

AsKC11, a Kunitz Peptide from Anemonia sulcata,Is a Novel Activator of G Protein-Coupled Inward-Rectifier Potassium Channels

(1) Background: G protein-coupled inward-rectifier potassium (GIRK) channels, especially neuronal GIRK1/2 channels, have been the focus of intense research interest for developing drugs against brain diseases. In this context, venom peptides that selectively activate GIRK channels can be seen as a new source for drug development. Here, we report on the identification and electrophysiological characterization of a novel activator of GIRK1/2 channels, AsKC11, found in the venom of the sea anemone Anemonia sulcata. (2) Methods: AsKC11 was purified from the sea anemone venom by reverse-phase chromatography and the sequence was identified by mass spectrometry. Using the two-electrode voltage-clamp technique, the activity of AsKC11 on GIRK1/2 channels was studied and its selectivity for other potassium channels was investigated. (3) Results: AsKC11, a Kunitz peptide found in the venom of A. sulcata, is the first peptide shown to directly activate neuronal GIRK1/2 channels independent from Gi/o protein activity, without affecting the inward-rectifier potassium channel (IRK1) and with only a minor effect on K_V1.6 channels. Thus, AsKC11 is a novel activator of GIRK channels resulting in larger K⁺ currents because of an increased chord conductance. (4) Conclusions: These discoveries provide new insights into a novel class of GIRK activators.     

Variability in the response toPseudomonas solanacearum of transgenic lines of potato carrying a cecropin gene analogue

Summary Transgenic potato plants of cv. Désirée carrying an antibacterial gene, coding for a cecropin lytic peptide analogue, were inoculated with a virulent strain ofPseudomonas solanacearum under controlled conditions. The disease index scored during three repeated infection trials indicated an increased variability in plant response among the transgenic lines which gave either a more susceptible or a more resistant response to the pathogen when compared with untransformed Désirée. Immunity toP. solanacearum was not observed, but it was possible to select a group of transgenic lines that showed resistance levels and disease development curves comparable to the field resistant cv. Cruza 148.     

大豆核孔蛋白GmNup96基因的克隆及生物信息学分析

从大豆品种天隆1号的叶片中克隆Gm Nup96基因的c DNA序列,对其编码的氨基酸序列、蛋白质理化性质、一级结构、二级结构、亚细胞定位等进行了生物信息学分析。结果表明:Gm Nup96基因编码1 022个氨基酸,为具有一定亲水能力的酸性蛋白,不具有信号肽,相对分子量为116.199 7 k Da;二级结构预测结果显示,Gm Nup96序列存在α-螺旋(46.87%)、无规则卷曲(26.32%)、延伸链(17.03%)和β-转角(9.78%),并无其它二级结构;系统进化树分析表明,大豆Gm Nup96基因与野生大豆、芸豆、绿豆、红小豆之间的亲缘关系更近。     

国审高产小麦扬辐麦9号选育及配套栽培技术

为进一步提高小麦品种扬辐麦4号的产量和综合抗性,采用辐射诱变与常规杂交育种技术相结合育成高产多穗抗倒小麦新品种扬辐麦9号(原代号:扬辐麦2149)。该品种2015—2016年度参加长江中下游冬麦组区域试验,平均产量6228.3 kg/hm²,比对照扬麦20增产7.49%,增产极显著;2016—2017年度续试,平均产量6454.1 kg/hm²,比对照扬麦20增产7.90%,增产极显著。2017—2018年参加国家长江中下游麦区生产试验,平均产量6385.8 kg/hm²,比对照增产6.72%。2019年通过国家审定(审定编号:国审麦20190005)。该品种分蘖性强,成穗率高,属多穗型小麦新品种,株高79.3 cm,株型较紧凑,穗层整齐,抗倒性强。高抗小麦黄花叶病,中抗赤霉病,中感纹枯病,高感白粉病、条锈和叶锈病,品质达中筋小麦。该品种的推广应用有利于进一步提高小麦产量水平,其综合抗性的提高有利于小麦清洁高效生产。新品种在推广应用过程中还需注意适期播种、优化群体结构、协调群体生长、合理运筹肥料提质增效、综合防治病虫害并及时收获。     

荔枝P型ATP酶基因家族鉴定及其在采后贮藏过程中响应拮抗菌N-1的表达模式分析

P型ATP酶是植物体内一种重要的膜转运蛋白,在果实能量代谢和酸代谢中发挥重要作用,并广泛参与植物离子运输、抗逆、细胞信号转导等各项生命活动,然而在荔枝或其他无患子科植物中尚未对P型ATP酶基因家族进行全面分析,本研究基于‘妃子笑’荔枝转录组数据,通过对这些基因进行生理生化分析、生物信息学分析和表达分析,共鉴定了28个P型ATP酶基因家族成员基因,探讨P型ATP酶在荔枝采后贮藏过程中的潜在功能。结构和系统发育分析表明该基因可以分为5个亚家族,同一亚族的基因在基因结构和motif基序方面具有很大的相似性,其蛋白二级结构主要以α-螺旋为主,亚细胞定位预测分析发现LcPAs多定位于线粒体（21/28）。通过转录组分析,在采后贮藏期间,荔枝P型ATP酶家族基因中上调表达的基因明显高于下调表达基因,暗示其可能在荔枝采后品质劣变过程中发挥着积极的抵御作用。使用拮抗菌N-1处理荔枝可以有效抑制其果实采后褐变和病害的发生,结合RT-qPCR结果,发现拮抗菌N-1处理能诱导LcPA1、LcPA7、LcPA8和LcPA19在整个贮藏过程中的上调表达,并维持了较高的ATP酶活性,进一步说明P型ATP酶家族基因在抑制荔枝病变过程中发挥着重要作用。本研究为了解荔枝P型ATP酶基因家族基因的进化和功能分析提供了参考,为深入了解拮抗菌N-1保鲜机理的分子调控机理提供新的证据。     

Screening and cDNA cloning of Kv1 potassium channel toxins in sea anemones

When 21 species of sea anemones were screened for Kv1 potassium channel toxins by competitive inhibition of the binding of (125)I-α-dendrotoxin to rat synaptosomal membranes, 11 species (two species of Actiniidae, one species of Hormathiidae, five species of Stichodactylidae and three species of Thalassianthidae) were found to be positive. Furthermore, full-length cDNAs encoding type 1 potassium channel toxins from three species of Stichodactylidae and three species of Thalassianthidae were cloned by a combination of RT-PCR, 3'RACE and 5'RACE. The precursors of these six toxins are commonly composed of signal peptide, propart and mature peptide portions. As for the mature peptide (35 amino acid residues), the six toxins share more than 90% sequence identities with one another and with κ(1.3)-SHTX-She1a (Shk) from Stichodactyla helianthus but only 34-63% identities with the other type 1 potassium channel toxins.