Glutamyl Oligopeptides as Factors Responsible for Tastes of a Proteinase-modified Soybean Protein

AK-toxin I, a host-specific toxin to Japanese pear (Pyrus serotina), was synthesized as its methyl ester from three precursor fragments: conjugated diene-carboxylic acid, chiral epoxyalcohol and β-methylphenylalanine. The epoxyalcohol fragment was derived from D-fructose, in which effective homologation of the hemiacetal carbon to alkyne by using dimethyl 1-diazo-2-oxopropylphosphonate was the key reaction. The diene-carboxylic acid fragment was prepared by repeated Wittig reactions, and was combined with the epoxyalcohol fragment by the Stille reaction. Esterification of the combined product with the stereochemically-pure β-methylphenylalanine fragment afforded the target compound. This method was used to prepare the methyl ester of tritium-labeled AK-toxin I with a specific radioactivity of 213 GBq/mmol.     

Cyanoacrylates. Herbicidal and Photosynthetic Inhibitory Activity

Various alkyl 3-phenylamino-2-cyano-and 2-ethoxycarbonylacrylates and 3-phenylamino-2-cyanoacrylonitriles were synthesized and assayed as inhibitors of the Hill reaction in isolated pea chloroplast suspensions and as pre-and post-emergent herbicides on mustard and barnyard grass. Many of the compounds were potent Hill reaction inhibitors and several showed significant herbicidal activity.     

炭疽水肿因子在大肠杆菌中高效表达及一步法纯化

【目的】本研究旨在建立一种简单快捷的炭疽水肿因子(EF)重组表达及纯化方法。【方法】构建GST-EF融合表达载体,基于EF基因的密码子使用偏好,选择菌株Escherichia coli BL21-Codon Plus(DE3)-RIL为表达宿主,对EF进行诱导表达;细胞透性技术分离粗蛋白,进而利用亲和层析一步法纯化EF;Native-PAGE、竞争性抑制实验及c AMP浓度分析用于鉴定EF的生物活性。【结果】实现了EF可溶性高效表达,透性化处理可有效抽提可溶性重组蛋白;利用亲和层析一步法纯化得到了纯度达96%的EF;EF可与保护性抗原(PA)结合形成水肿毒素,该毒素能够急剧提高CHO-K1细胞中c AMP的浓度。【结论】本研究建立了一种高效快速制备具有生物活性的炭疽水肿因子的方法,为炭疽相关研究工作提供了新的选择。     

CMG2-Fc 融合蛋白突变体筛选及中和炭疽毒素活性分析

目的:筛选能有效中和炭疽毒素和抵抗炭疽毒素损伤细胞的CMG2-Fc(炭疽毒素受体II-人免疫球蛋白Fc段融合蛋白)突变体。方法:运用FoldX等计算软件分析CMG2与PA晶体学结构,设计能提高CMG2-PA亲和力的突变体分子,并与人IgG1Fc片段构成融合基因,转染CHO-S细胞并通过亲和层析获得CMG2-Fc突变体蛋白,通过亲和力检测和细胞保护实验分析各突变体中和炭疽毒素能力。结果:筛选并表达了8个CMG2-Fc突变体分子,亲和力实验显示其中E117Q突变可明显提高CMG2-Fc与PA的亲和力(KD=1.35×10-11 mol/L),细胞保护实验提示E117Q突变能有效提高CMG2-Fc中和炭疽毒素能力(CMG2-Fc(E117Q)的IC50为15 ng/μL,而wt CMG2-Fc的IC50为50ng/μL)。结论:CMG2-Fc(E117Q)突变体分子可作为拮抗炭疽毒素损伤的炭疽治疗药物分子,进行进一步研究。     

高通量血液透析治疗慢性肾衰竭尿毒症的疗效及对尿毒症毒素、免疫球蛋白及肺功能指标的影响

目的:探讨高通量血液透析(HFHD)治疗慢性肾衰竭尿毒症的疗效及对尿毒症毒素、免疫球蛋白及肺功能指标的影响。方法:选取90例于2012年1月-2017年3月期间在喀什地区第一人民医院治疗的慢性肾衰竭尿毒症患者,依据随机数字表法将其分为对照组(n=45)和观察组(n=45),对照组给予血液透析滤过(HDF)治疗,观察组给予HFHD治疗,两组均透析治疗1个月。对比两组患者透析前后症状缓解情况及尿毒症毒素、免疫球蛋白及肺功能指标水平,记录两组相关并发症的发生情况。结果:透析治疗结束后观察组患者缓解率为91.11%(41/45),高于对照组的73.33%(33/45)(P0.05)。两组患者透析后血磷(P~-)、血钾(K~+)、甲状旁腺激素(PTH)、β2-微球蛋白(β2-MG)水平明显低于透析前,血钙(Ca~(2+))水平明显高于透析前(P0.05);观察组透析后K~+、Ca~(2+)、P~-等尿毒症毒素水平与对照组比较差异无统计学意义(P0.05),观察组透析后PTH、β2-MG水平明显低于对照组(P0.05)。透析后,两组患者的免疫球蛋白Ig M、Ig A、Ig G水平均较透析前上升,且观察组高于对照组(P0.05)。透析后,两组患者残气量(RV)均低于治疗前,最大肺活量(FVC)、肺活量(VC)、肺总量(TLC)均高于治疗前,且观察组RV低于对照组,FVC、VC、TLC均高于对照组(P0.05)。观察组并发症发生率为8.89%(4/45),低于对照组的24.44%(11/45)(P0.05)。结论:HFHD治疗慢性肾衰竭尿毒症能够安全有效地清除尿毒症毒素,缓解患者的临床症状,且能够提高患者的免疫功能和肺功能。     

Multi-species okadaic acid contamination and human poisoning during a massive bloom of Dinophysis acuminata complex in southern Brazil

On June 2016, a major bloom of Dinophysis acuminata complex was noticed over the coast of Paraná State (PR), southern Brazil, an area unprotected by any official monitoring program. Here we report the results of an extensive sampling effort that ultimately led PR authorities to issue the first State shellfish-harvesting ban due to multi-species okadaic acid (OA) contamination. During its peak, the bloom covered an area of 201 km² (∼2.0–3.5 × 54.0 km), attaining unprecedentedly high cell densities along the shallow (<15 m) continental shelf (mean 2.2 × 10⁵, maximum 2.1 × 10⁶ cells L⁻¹) and adjacent sandy beaches (mean 2.8 × 10⁵, maximum 5.2 × 10⁶ cells L⁻¹). Only OA was detected in suspension (max. 188 ng L⁻¹). Toxin levels measured in bivalves were several times greater than the regulatory limit of 160 ng g⁻¹, reaching up to 3600 ng g⁻¹ in Crassostrea gasar, by far the highest OA concentrations ever reported in oysters worldwide, 7700 ng g⁻¹ in brown mussels, Perna perna, and lower levels in clams, Anomalocardia brasiliana, and mangrove mussels, Mytella spp. Nine cases of human intoxication were officially reported and five people were hospitalized with typical symptoms of Diarrhetic Shellfish Poisoning linked to the consumption of contaminated bivalves. All bivalves quickly converted most of the OA into its esterified form, DTX-3, and eliminated the toxins only a few weeks following the bloom, with C. gasar being the slowest-detoxifying species. Lower OA levels were accumulated in zooplankton, gastropods and several novel toxin vectors, including benthic organisms such as sand dollars Mellita quinquiesperforata and the ghost-shrimp Callichirus major, which may act as a good indicator of the presence of toxins in sandy beaches, and pelagic fish species that can serve as potential alternative sources of OA to humans (Chaetodipterus faber and Mugil liza). Monitoring toxin contamination in seafood other than bivalves is thus recommended to ensure comprehensive human health protection during massive Dinophysis blooms. Additionally, since OA was also present at low concentrations in the liver of Guiana dolphins Sotalia guianensis and penguins Spheniscus magellanicus, exposure to biotoxins should be considered in conservation actions involving threatened and near-threatened marine organisms in this region.     

Trends in Dinophysis abundance and diarrhetic shellfish toxin levels in California mussels (Mytilus californianus) from Monterey Bay,California

Diarrhetic shellfish toxins (DSTs) are produced by the marine dinoflagellate, Dinophysis, as well as select species of benthic Prorocentrum. The DSTs can bioaccumulate in shellfish and cause gastrointestinal illness when humans consume high levels of this toxin. Although not routinely monitored throughout the U.S., recent studies in Washington, Texas, and New York suggest DSTs may be widespread throughout U.S. coastal waters. This study describes a four-year time series (2013–2016) of Dinophysis concentration and DST level in California mussels (Mytilus californianus) from Santa Cruz Municipal Wharf (SCMW) in Monterey Bay, California. Results show a maximum Dinophysis concentration of 9404 cells/L during this study and suggest Dinophysis persists as a member of the background phytoplankton community throughout the year. In California mussels, DSTs were found at persistent low levels throughout the course of this study, and exceeded the FDA guidance level of 160 ng/g 19 out of 192 weeks sampled. Concentrations of Dinophysis alone are a positive but weak predictor of DST level in California mussels, and basic environmental variables (temperature, salinity, and nutrients) do not sufficiently explain variation in Dinophysis concentration at SCMW. This study demonstrates that Dinophysis in Monterey Bay are producing DSTs that accumulate in local shellfish throughout the year, occasionally reaching levels of concern.     

Killer toxin-like chitinases in filamentous fungi: Structure,regulation and potential roles in fungal biology

Fungal chitinases are hydrolytic enzymes responsible for degradation of chitin. Chitinases are involved in several aspects of fungal biology, including cell wall remodelling during hyphal growth, conidial germination, autolysis, mycoparasitism and nutrient acquisition. They are divided into three distinct phylogenetic groups; A, B and C. Chitinases from the C group show structural similarities with the killer toxin zymocin produced by the yeast Kluyveromyces lactis and it is speculated that they have a similar function in filamentous ascomycetes, by facilitating penetration of toxins into cells of competing individuals. Genome analyses show that certain fungal species with a mycoparasitic lifestyle contain high numbers of killer toxin-like chitinases, compared with specialized saprotrophs and plant pathogens. Recent developments within this research field have revealed considerable variation in the modular structure and regulation of killer toxin-like chitinases, suggesting more diverse roles than merely fungal-fungal interactions. In this review, we summarize the current knowledge about this intriguing class of chitinases, including their modular structure, evolution, gene regulation, and functional analyses in mycoparasitic as well as in saprotrophic species. We also propose important questions for future research.     

Identification of a α‐helical molten globule intermediate and structural characterization of β‐cardiotoxin,an all β‐sheet protein isolated from the venom of Ophiophagus hannah (king cobra)

β‐Cardiotoxin is a novel member of the snake venom three‐finger toxin (3FTX) family. This is the first exogenous protein to antagonize β‐adrenergic receptors and thereby causing reduction in heart rates (bradycardia) when administered into animals, unlike the conventional cardiotoxins as reported earlier. 3FTXs are stable all β‐sheet peptides with 60–80 amino acid residues. Here, we describe the three‐dimensional crystal structure of β‐cardiotoxin together with the identification of a molten globule intermediate in the unfolding pathway of this protein. In spite of the overall structural similarity of this protein with conventional cardiotoxins, there are notable differences observed at the loop region and in the charge distribution on the surface, which are known to be critical for cytolytic activity of cardiotoxins. The molten globule intermediate state present in the thermal unfolding pathway of β‐cardiotoxin was however not observed during the chemical denaturation of the protein. Interestingly, circular dichroism (CD) and NMR studies revealed the presence of α‐helical secondary structure in the molten globule intermediate. These results point to substantial conformational plasticity of β‐cardiotoxin, which might aid the protein in responding to the sometimes conflicting demands of structure, stability, and function during its biological lifetime.     

A Centipede Toxin Family Defines an Ancient Class of CSαβ Defensins

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