构建基于CRISPR/Cas9技术敲除ALOX5的质粒

旨在利用CRISPR/Cas9技术构建敲除花生四烯5-脂氧合酶基因(Arachidonate 5-lipoxygenase gene,ALOX5)的重组质粒。设计合成3对靶向敲除ALOX5第六外显子的sgRNA,将其分别插入到CRISPR/Cas9质粒骨架pX458载体中,转化感受态大肠杆菌DH5α后挑取克隆,通过测序评估重组质粒是否构建成功。将构建好的重组质粒转染293T细胞,在荧光显微镜下观察转染效果,挑取转染成功的细胞,用试剂盒提取转染细胞基因组DNA,PCR扩增含敲除位点的DNA片段,用测序技术获得核苷酸序列,用DNAStar软件分析转染细胞中ALOX5基因敲除情况。测序结果表明2对双链sgRNA寡核苷酸已插入质粒,且序列正确,靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5构建成功。其在293T细胞中的转染效率约为50%,用一代测序法未检测到sgRNAs的切割效果。初步表明利用CRISPR/Cas9技术成功构建靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5。     

The effect of the nature of the iron sources on the growth of different Corynebacterium diphtheriae strains

In the study of the influence of organic and inorganic sources of iron on the growth of 5 C. diphtheriae clinical isolates bacterial growth was found to depended on the nature of the source of iron and its concentration. Differences between the strains in the level of growth, observed when ferric sulfate was used as the only source of iron in the medium, were established. Quantitative differences in the concentrations of inorganic and organic sources of iron, necessary for growth, were determined. The influence of three chemical chelators on the growth of C. diphtheriae under the conditions of iron deficiency in the culture medium was studied. The results of the study are indicative of the possibility of the differentiation of C. diphtheriae isolated according to the level of iron consumption.     

杨树溃疡病生防菌株N6-34抗菌活性物质的分离纯化与结构鉴定

【背景】杨树溃疡病是一种主要由葡萄座腔菌引起的杨树枝干病害,危害严重。前期从杨树中分离到一株内生拮抗细菌N6-34,研究表明该菌株拮抗效果好,对多种植物病原菌均有较强的拮抗作用。【目的】对拮抗细菌N6-34产生的抗菌活性物质进行分离纯化,并鉴定了活性物质组分的结构。【方法】通过硫酸铵盐析、甲醇抽提、分子筛、高效液相色谱等方法分离纯化N6-34菌株的抗菌活性物质,并对其进行结构鉴定。【结果】N6-34菌株发酵液经多步分离纯化,共获得14个组分,其中有13个组分具有抗菌活性,经一级质谱分析,获得了13种抗菌活性组分的分子量;经二级质谱分析,将13种抗菌活性物质鉴定为Fengycin A或Fengycin B的同系物或同分异构体。【结论】从N6-34菌株发酵液中分离获得了13种抗菌成分,为杨树溃疡病的生物防治提供了理论依据。     

Laccase-based biosensor for determination of polyphenols: determination of catechols in tea.

A new method of amperometric determination of phenolic compounds using an enzyme electrode is proposed. The latter represents the combination of the oxygen electrode and immobilized laccase. Analytical systems of flow injection and batch types were considered. A method of immobilization was developed that provided an increase in the stability of the enzyme. Optimal conditions for biosensor operation were found. The time needed for analysis in the flow injection mode was below 100 s. A column with immobilized enzyme could be used for up to 500 determinations of phenolic compounds without decrease of the enzyme activity. The practical validity of the method was demonstrated by tannin analysis in tea of different brands.     

The antiradiation activity and mechanism of action of dimethylaminodithiazines

A new group of radioprotective agents, dimethylaminodithiazines, was discovered. The agents are effective when used in relatively small amounts and exert a radioprotective effect rapidly with both intraperitoneal and per os injections. The role of inhibition of oxidative processes and DNA biosynthesis in the mechanism of radioprotective action of dimethylaminodithiazines is discussed.     

Antitumor Activity of Probiotic Bacteria and the Bacterial Association in the Tumor Growth Model and in Cell Culture

Strains of lactic acid and propionic acid bacteria and the A-5 bacterial association composed of the Lactobacillus brevis B-3/A-26, L. plantarum 2v/А-6, L. plantarum 14d/I, and Propionibacterium shermanii 2/10 cultures in equal proportion exhibiting high antitumor activity have been selected using the fungus Fusarium bulbigenum var. blasticola tumorlike growth model. L. acidophilus 27w, L. plantarum 2v/I, 2v/A-6, and 22A-310, L. fermentum 127A-4, L. cellobiosus 7n, L. salivarius 39n, P. shermanii 2/10, and the А-5 association were shown to inhibit completely the tumorlike growth of the fungus when present as 2% native cultures in the nutrient medium. The high antitumor activity of the A-5 association comparable with that of the therapeutic agents doxorubicin and cyclophosphamide was confirmed using human tumor cell lines originating from different organs (HepG2, RD, and H9). It has been demonstrated that the bacterial strains comprising the A-5 association were resistant to doxorubicin and methotrexate, the antitumor drugs tested.     

Molecular evolution of cytochrome c oxidase: rate variation among subunit VIa isoforms

Cytochrome c oxidase (COX) consists of 13 subunits, 3 encoded in the mitochondrial genome and 10 in the nucleus. Little is known of the role of the nuclear-encoded subunits, some of which exhibit tissue-specific isoforms. Subunit VIa is unique in having tissue-specific isoforms in all mammalian species examined. We examined relative evolutionary rates for the COX6A heart (H) and liver (L) isoform genes along the length of the molecule, specifically in relation to the tissue-specific function(s) of the two isoforms. Nonsynonymous (amino acid replacement) substitutions in the COX6AH gene occurred more frequently than in the ubiquitously expressed COX6AL gene. Maximum-parsimony analysis and sequence divergences from reconstructed ancestral sequences revealed that after the ancestral COX6A gene duplicated to yield the genes for the H and L isoforms, the sequences encoding the mitochondrial matrix region of the COX VIa protein experienced an elevated rate of nonsynonymous substitutions relative to synonymous substitutions. This is expected for relaxed selective constraints after gene duplication followed by purifying selection to preserve the replacements with tissue-specific functions.      

Effect of the dielectric constant of the medium on the photooxidation of chlorophyll a by parabenzoquinone]

Spectrophotometric study was carried out of the effect of medium polarity on the parabenzoquinone photooxidation of chlorophyll "a" at lower temperature using as solvents toluene and CCl4 containing different quantities of polaric solvents, methanole, ethanole, amilic alkohol, acetone. It is shown that the photoreaction with the formation of primary photooxidized form of the pigment starts only if some amount of the polar component is in the medium. The data are in favour of the suggestion that chlorophyll and quinone in non-polar medium form an reaction complex which disintegrates under the given conditions at a definite value of medium polarity.