Isolation of the S-peptide formed on digestion of fructose 1,6-bisphosphatase with subtilisin and its non-covalent association with the enzyme protein.

Digestion of rabbit liver fructose 1,6-bisphosphatase with subtilisin results in a several-fold increase in catalytic activity measured at pH 9.2. This change is due to cleavage of a peptide bond located 60 amino acid residues from the NH₂-terminus. The S-peptide and the residual subunit appear as separate peptides in sodium dodecyl sulfate polyacrylamide gel electrophoresis and the S-peptide can be isolated by gel filtration in 9% HCOOH. Under nondissociating conditions, however, the S-peptide remains associated with the protein, and the tetrameric structure and original molecular weight are preserved. Thus the nicking of the peptide chain by subtilisin causes a conformation change that alters the catalytic properties of the enzyme.     

Reconstitution of biological molecular generators of electric current. Cytochrome oxidase.

1. Direct measurement of the electric current generation by cytochrome oxidase has been carried out. To this end, two procedures were used. The simpler one consists in formation of planar artificial membrane from the mixture of decane solution of soya bean phospholipids and beef heart cytochrome oxidase. Addition of cytochrome c and ascorbate to one of the two compartments separated by the cytochrome oxidase-containing planar membrane was found to result in a transmembrane electric potential difference being formed (plus on cytochrome c side of the membrane). Maximal values of potential differences obtained by this method were about 40 mV. Much higher potentials were observed when another ("photeoliposome-planar membrane") method was applied. In this case cytochrome oxidase was reconstituted with phospholipid to form proteoliposomes which adhered to planar phospholipid membrane in the presence of Ca2+ ions. Addition of cytochrome c and ascorbate to the proteoliposome-containing compartment gives rise to generation of an electric potential difference across the planar membrane, which reached 100 mV at a current of about 1 X 10(-11) A (minus in the proteoliposome-free compartment). The electromotive force of this generator was estimated as being about 0.2 V. If ascorbate and proteoliposomes were added into different compartments, a penetrating hydrogen atom carrier (phenazine methosulfate, (PMS) or tetramethyl-p-phenylenediamine (TMPD)) was required for a membrane potential to be formed. Generation of an electric potential difference of the opposite direction (plus in the proteoliposome-free compartment) was revealed in experiments with cytochrome oxidase proteoliposome containing cytochrome c in their interior. In this case, addition of PMS or TMPD was necessary. 2. In the suspension of cytochrome oxidase proteoliposome the uptake of a cationic penetrant (tetraphenyl phosphonium cation) was found to be coupled with electron transfer via external cytochrome c. Electron transfer via intraproteoliposomal cytochrome c induced the uptake of anionic penetrants (tetraphenyl borate and phenyldicarbaundecaborane anions). 3. All the above effects were sensitive to cyanide and protonophorous uncouplers. 4. In proteoliposomes containing both cytochrome oxidase and bacteriorhodopsin, the light- and oxidation-dependent generations of membrane potential have been revealed. 5. The data obtained are in agreement with Mitchell's idea of transmembrane electron flow in the cytochrome oxidase segment of the respiratory chain.     

The ultrastructure of the middle cerebral artery and its associated nerve fibers in the squirrel monkey and baboon

This study describes the ultrastructural characteristics of the middle cerebral artery and its related neural elements in the squirrel monkey and baboon. The cytoarchitecture of the M-1 segment as well as that of the smaller extracerebral and intracerebral vessels is comparable in both animals.Smooth muscle elements are occasionally found within the intimai lining. The nerve bundles associated with vessels contain fewer myelinated fibers as the vessel diameter decreases. The cytological relationship between the neural structures and the smooth muscle cells are discussed.     

滇西北退化高寒草甸植物群落结构对刈割的响应

为探究滇西北不同退化级别高寒草甸植物群落结构对外界干扰的响应敏感性,以香格里拉市的典型高寒草甸为研究对象,于2018-2020年在三个退化梯度上（严重退化,S1;中度退化,S2;轻度退化,S3）开展控制刈割实验,进而分析草甸植物物种丰富度、群落组成相似性、群落复杂度和关键种的变化规律。结果表明：（1）刈割后,S1的物种丰富度显著增加（P<0.05）,S2和S3的物种丰富度未发生显著变化（P>0.05）;（2）相较于S2和S3,S1梯度的植物群落组成变化最大;（3） S1、S2和S3的植物群落复杂度在刈割后均呈先下降后增加的趋势,但S1的植物群落复杂度变化幅度高于S2和S3;（4）刈割导致各退化草甸植物群落的关键种发生了变化,2018、2019和2020年S1梯度的关键种在豆科（Leguminsae）和蔷薇科（Rosaceae）之间变化,S2梯度的关键种在禾本科（Gramineae）和菊科（Compositae）之间变化,S3梯度的关键种在蔷薇科（Rosaceae）、菊科（Compositae）和禾本科（Gramineae）之间转变。研究表明,滇西北高寒草甸植物物种丰富度、群落组成和群落复杂度对外界干扰响应的敏感性可能随退化加剧而上升,但群落关键种的响应过程较复杂。     

微塑料对稻田土壤温室气体排放和微生物群落的影响

微塑料因在土壤环境中广泛存在及其潜在的生态风险而受到越来越多的关注。微塑料的赋存会改变土壤理化性质,并对土壤微生物群落及其驱动的生物地球化学过程产生影响,而相关研究尚处于起步阶段。可生物降解塑料作为传统塑料的替代品,越来越多地应用于农业活动,并释放到土壤中。然而,可生物降解微塑料对土壤微生物特性产生影响的研究鲜有报道。基于此,本试验以我国三江平原水稻田土壤为研究对象,选取了2种常见的微塑料为试验材料,分别为传统型微塑料聚丙烯(Polypropylene,PP)和可降解微塑料聚乳酸(Polylactic acid,PLA),进行了为期41d的微宇宙培养实验,旨在分析不同浓度与类型的微塑料对土壤可溶性有机碳(Dissolved Organic Carbon,DOC)含量及官能团特征、温室气体排放以及微生物群落结构的差异性影响。结果表明,传统型微塑料PP与可降解微塑料PLA添加均对土壤理化性质与微生物群落产生显著影响。其中,微塑料添加大体上增加了土壤DOC含量,PLA的促进作用较为明显,且增加含量与微塑料添加量呈正相关;PP和PLA均影响土壤DOC分子结构,削弱了土壤团聚化程度并促进了大分子量DOC化合物的生成;微塑料的添加促进土壤CH₄排放,而有效抑制了土壤CO₂排放;微塑料显著改变了土壤细菌和真菌群落的丰富度与多样性。相关分析结果表明,土壤CO₂累计排放量与芳香族化合物结构及疏水性等官能团特征、变形菌门(Proteobacteria)与放线菌门(Actinobacteria)均呈显著正相关关系。以上结果表明,微塑料添加改变了土壤DOC含量及官能团特征与微生物环境,进而影响土壤温室气体排放。本研究为今后微塑料对土壤地球化学和微生物特性的影响研究提供了科学的思路,同时也有助于评估微塑料对土壤生态系统的生态风险。     

Breeding of High Daptomycin-Producing Strain by Streptomycin Resistance Superposition

Daptomycin is a cyclolipopeptide antibiotic produced by Streptomyces roseosporus. It is widely used to treat drug-resistant bacterial infections; however, daptomycin yield in wild strains is very low. To improve the daptomycin production by the strain BNCC 342432, a modified method of ribosome engineering with superposition of streptomycin resistance was adopted in this study. The highest-yield mutant strain SR-2620 was obtained by increasing streptomycin resistance of BNCC 342432, and achieved daptomycin production of 38.5 mg/l in shake-flask fermentation, 1.79-fold higher than the parent strain and its heredity stability was stable. The morphological characteristics of the two strains were significantly different, and the 440^th base G of the rpsL gene in the mutant strain was deleted, which resulted in a frameshift mutation. Our results demonstrate that gradually increasing strain resistance to streptomycin was an effective breeding method to improve daptomycin yield in S. roseosporus. Open in a separate window     

黄河三角洲芦苇湿地底栖无脊椎动物与环境因子的关系研究——以石油开采区与淡水补给区为例

分别于2018年5月和8月对黄河三角洲芦苇湿地19(淡水补给区11处,石油开采区8处)处采样点的底栖无脊椎动物和水体理化指标进行调查采样,运用统计方法分析两个区域物种组成、优势种、多样性、群落结构以及与环境因子的关系。结果表明:两季共采集到底栖无脊椎动物54种,主要以水生昆虫、腹足纲和软甲纲为主,淡水补给区和石油开采区各类群组成差异明显。独立样本T检验表明淡水补给区和石油开采区水体理化指标间差异显著(P<0.05)。双因子方差分析显示,昆虫纲和腹足纲密度在两区域差异显著(P<0.05),软甲纲和腹足纲密度在季节上差异明显(P<0.05)。底栖无脊椎动物优势种共10种,淡水补给区指示物种8种,而石油开采区未发现有指示物种。聚类和非参数多维排序(nM DS)显示,底栖无脊椎动物群落结构相似性较低; RDA结果表明:淡水补给区底栖无脊椎动物群落结构主要受Cond,TDS,Sal,pH,Eh,HCO₃^-,SO₄^2-等环境因子的影响。石油开采区底栖无脊椎动物群落结构影响较大的环境因子为HCO...     

新疆棉花黄萎病菌拮抗细菌的分离、筛选与鉴定

【背景】棉花黄萎病是由大丽轮枝菌(Verticillium dahliae Kleb.)引起的一种世界性病害,近年来对该病害的生物防治因具有环境友好和人畜安全的特性而倍受关注。【目的】筛选棉花黄萎病高效拮抗细菌并对其进行鉴定,为棉花黄萎病的生物防治扩充菌种资源。【方法】采用稀释涂布平板法分离细菌,并进行拮抗细菌的初筛和复筛,通过形态特征、生理生化特征和16S rRNA基因序列分析对筛选到的细菌进行鉴定,确定其分类地位。【结果】初筛分离到535株对病原菌具有拮抗作用的细菌,并选取了108株拮抗细菌进行复筛,最终筛选到了4株优势拮抗细菌。通过形态观察、生理生化特征和16SrRNA基因序列分析,将菌株BHZ-29、SHT-15、SHZ-24和SMT-24分别鉴定为贝莱斯芽孢杆菌(Bacillusvelezensis)、枯草芽孢杆菌斯皮兹仁亚种(Bacillus subtilis subsp. spizizenii)、萎缩芽孢杆菌(Bacillus atrophaeus)和香草芽孢杆菌(Bacillus vanillea)。【结论】获得了4株高效拮抗细菌,并且首次报道了香草芽孢杆菌对棉花黄萎病菌具有抑制作用。     

The mushroom ribosome-inactivating protein lyophyllin exerts deleterious effects on mouse embryonic development in vitro

Earlier investigations disclose that some plant ribosome-inactivating proteins (RIPs) adversely affect mouse embryonic development. In the present study, a mushroom RIP, namely lyophyllin from Lyophyllum shimeji, was isolated, partially sequenced, and its translation inhibitory activity determined. Its teratogenicity was studied by using a technique entailing microinjection and postimplantation whole-embryo culture. It was found that embryonic abnormalities during the period of organogenesis from E8.5 to E9.5 were induced by lyophyllin at a concentration as low as 50 μg/ml, and when the lyophyllin concentration was raised, the number of abnormal embryos increased, the final somite number decreased, and the abnormalities increased in severity. The affected embryonic structures included the cranial neural tube, forelimb buds, branchial arches, and body axis, while optic and otic placodes were more resistant. Lyophyllin at a concentration higher than 500 μg/ml also induced forebrain blisters within the cranial mesenchyme. When the abnormal embryos were examined histologically, an increase of cell death was found to be associated with abnormal structures, indicating that cell death may be one of the underlying causes of teratogenicity of the mushroom RIP. This constitutes the first report on the teratogenicity of a mushroom RIP.     

An intramolecular disulfide bond is required for the thermostability of methyl parathion hydrolase, OPHC2

OPHC2, a methyl parathion hydrolase (MPH) from Pseudomonas pseudoalcaligenes C2-1 (CGMCC 1150), can degrade a wide range of organophosphate pesticides. Compared with other MPHs, OPHC2 exhibits high thermostability. Its thermostability mechanism, however, remains unknown. In the present study, sequence analysis demonstrated that two cysteines (Cys110 and Cys146) exist in OPHC2, but not in other MPHs. The three-dimensional structural model of OPHC2 performed by computer-assisted homology modelling revealed a potential stacking network with residues Cys110 and Cys146, which probably formed an intramolecular disulfide bond. Furthermore, both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and thiol-titration analyses indicated that OPHC2 contains a disulfide bond. Substitution of the disulfide bond-forming cysteines with alanine, leucine or methionine residues substantially decreased the thermostability of OPHC2, suggesting that disulfide bond formation affects conformational stability. These results, combined with three-dimensional structural modelling, demonstrated that the formation of a C110-C146 disulfide bond may stabilise the conformation of OPHC2, contributing to its thermostability.