Effects of the epoxide hydrase inhibitor, 1,1,1-trichloropropane-2,3-oxide on the genetic activity of aflatoxin B1 metabolites in in vitro activation test systems.

The epoxide hydrase inhibitor 1,1,1-trichloroprophane-2,3-oxide (TCPO) was genetically active to cells of S. cerevisiae and conidia of N. crassa. This genetic activity could be eliminated or reduced to near spontaneous levels in the presence of the S-9 fraction of hamster liver homogenate. The addition of TCPO to an in vitro activation system containing aflatoxin B1 resulted in an increase in the genetic activity of aflatoxin B1, and this increase was dependent on the dose of TCPO. These results are discussed in relation to the possible metabolism of the promutagen aflatoxin B1.     

Use of the spot, plate and suspension test systems for the detection of the mutagenicity of environmental agents and chemical carcinogens in Neurospora crassa.

N23 and N24, selected from hundreds of ad-3 mutants, have been used as testers for the spot, plate and suspension tests in Neurospora crassa. These two testers are highly sensitive to mutagens and are revertible by a specific group of chemicals. N23 can be reverted from an adenine-dependence to adenine-independence by agents which cause base-pair substitutions whereas N24 can be reverted by frameshift mutagens. Studies described here show that spot, plate and suspension tests using testers N23 and N24 are satisfactory substitutes for the ad-3 forward-mutation system for screening the mutagenic activity of environmental agents and chemical carcinogens in N. crassa.     

Regulation of Noradrenaline Release from Rat Occipital Cortex Tissue Chops by α2-Adrenergic Agonists

Noradrenaline (NA) and the alpha 2-adrenergic agonists clonidine, BHT-920, and UK 14304-18 inhibit potassium-evoked release of [3H]NA from rat occipital cortex tissue chops with similar potencies. NA (10(-5) M) was most effective as up to 85% inhibition could be observed compared with 75%, 55%, and 35% for UK 14304-18, clonidine, and BHT-920, respectively, all at 10(-5) M. Potassium-evoked release was enhanced by both forskolin (10(-5) M) and 1 mM dibutyryl cyclic AMP. Pretreatment of tissue chops with 1 mM dibutyryl cyclic AMP in the presence of 3-isobutyl-1-methylxanthine partially reversed the alpha 2-adrenergic agonist inhibition of NA release. No reversal of inhibition was observed following pretreatment with 10(-5) M forskolin. The effects of clonidine, BHT-920, UK-14308-18, and NA on cyclic AMP formation stimulated by (a) forskolin, (b) isoprenaline, (c) adenosine, (d) potassium, and (e) NA were examined. Only cAMP formation stimulated by NA was inhibited by these alpha 2-adrenergic agonists. These results suggest that only a small fraction of adenylate cyclase in rat occipital cortex is coupled to alpha 2-adrenergic receptors. These results are discussed in relation to recent findings that several alpha 2-adrenergic receptor subtypes occur, not all of which are coupled to the inhibition of adenylate cyclase, and that alpha 2-adrenergic receptors inhibit NA release in rat occipital cortex by a mechanism that does not involve decreasing cyclic AMP levels.     

Molecular variants of human papillomavirus type 16 from four continents suggest ancient pandemic spread of the virus and its coevolution with humankind.

We have amplified by the polymerase chain reaction, cloned, and sequenced genomic segments of 118 human papillomavirus type 16 (HPV-16) isolates from 76 cervical biopsy, 14 cervical smear, 3 vulval biopsy, 2 penile biopsy, 2 anal biopsy, and 1 vaginal biopsy sample and two cell lines. The specimens were taken from patients in four countries--Singapore, Brazil, Tanzania, and Germany. The sequence of a 364-bp fragment of the long control region of the virus revealed 38 variants, most of which differed by one or several point mutations. Phylogenetic trees were constructed by distance matrix methods and a transformation series approach. The trees based on the long control region were supported by another set based on the complete E5 protein-coding region. Both sets had two main branches. Nearly all of the variants from Tanzania were assigned to one (African) branch, and all of the German and most of the Singaporean variants were assigned to the other (Eurasian) branch. While some German and Singaporean variants were identical, each group also contained variants that formed unique branches. In contrast to the group-internal homogeneity of the Singaporean, German, and Tanzanian variants, the Brazilian variants were clearly divided between the two branches. Exceptions to this were the seven Singaporean isolates with mutational patterns typical of the Tanzanian isolates. The data suggest that HPV-16 evolved separately for a long period in Africa and Eurasia. Representatives of both branches may have been transferred to Brazil via past colonial immigration. The comparable efficiencies of transfer of the African and the Eurasian variants to the New World suggest pandemic spread of HPV-16 in past centuries. Representatives of the African branch were possibly transferred to the Far East along old Arab and Indonesian sailing routes. Our data also support the view that HPV-16 is a well-defined virus type, since the variants show only a maximal genomic divergence of about 5%. The small amount of divergence in any one geographic location and the lack of marked divergence between the Tanzanian and Brazilian African genome variants two centuries after their likely introduction into the New World suggest a very slow rate of viral evolution. The phylogenetic tree therefore probably represents a minimum of several centuries of evolution, if not an age equal to that of the respective human races.     

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. IV. Comparison of dose-response curves for MNNG, 4NQO and ICR-170 induced inactivation and mutation-induction

The genetic effects of MNNG, 4NQO and ICR-170 have been compared on 5 different UV-sensitive strains and a standard wild-type strain of Neurospora crassa with regard to inactivation and the induction of forward-mutations at the ad-3A and ad-3B loci. Whereas all UV-sensitive strains (upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) are more sensitive to inactivation by MNNG and ICR-170 than wild-type, only uvs-5 shows survival comparable to wild-type after 4NQO treatment, all other strains are more sensitive to 4NQO. In contrast to the effects on inactivation, a wide variety of effects were found for the induction of ad-3A and ad-3B mutations: higher forward-mutation frequencies than were found in wild-type were obtained after treatment with MNNG or 4NQO for upr-1 and uvs-2, no significant increase over the spontaneous mutation frequency was found with uvs-3 after MNNG, 4NQO or ICR-170 treatment; mutation frequencies comparable to that found in wild-type were obtained with uvs-6 after MNNG, 4NQO or ICR-170 treatment and with upr-1 after ICR-170 treatment. Lower forward-mutation frequencies than were found in wild-type were obtained with uvs-2 after ICR-170 treatment and with uvs-5 after MNNG, 4NQO or ICR-170 treatment. These data clearly show that the process of forward-mutation at the ad-3A and ad-3B loci is under genetic control by mutations at other loci (e.g. upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) and that the effect is markedly mutagen-dependent.     

Gel electrophoresis patterns of proteins and peroxidases of excised tomato cotyledons subjected to mannitol induced water stress

Excised tomato cotyledonswere subjected to mannitol induced water stress solutions for three days. Extracts of proteins and the enzyme peroxidase were made and separated with polyacrylamide gel electrophoresis. The water stress injury caused increases in bigger protein molecules but decreases in smaller protein molecules. The small fastest moving peroxidase isozyme was almost completely eliminated. Applications of growth regulators to the stress solutions indicated that the protein and peroxidase changes could be explained on the basis of reduction in endogenous cytokinin activities by the water stress.     

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。     

Neural induction: Historical views and application to pluripotent stem cells

Embryonic stem (ES) cells are a useful experimental material to recapitulate the differentiation steps of early embryos, which are usually invisible and inaccessible from outside of the body, especially in mammals. ES cells have greatly facilitated the analyses of gene expression profiles and cell characteristics. In addition, understanding the mechanisms during neural differentiation is important for clinical purposes, such as developing new therapeutic methods or regenerative medicine. As neurons have very limited regenerative ability, neurodegenerative diseases are usually intractable, and patients suffer from the disease throughout their lifetimes. The functional cells generated from ES cells in vitro could replace degenerative areas by transplantation. In this review, we will first demonstrate the historical views and widely accepted concepts regarding the molecular mechanisms of neural induction and positional information to produce the specific types of neurons in model animals. Next, we will describe how these concepts have recently been applied to the research in the establishment of the methodology of neural differentiation from mammalian ES cells. Finally, we will focus on examples of the applications of differentiation systems to clinical purposes. Overall, the discussion will focus on how historical developmental studies are applied to state‐of‐the‐art stem cell research.     

羌活与宽叶羌活的群体遗传结构和种间分化研究

为了合理利用羌活和宽叶羌活的药用植物资源,同时保护其物种多样性,该研究利用SSR分子标记技术对羌活与宽叶羌活邻域及异域分布的23个自然种群,共计227个个体进行多样性和种间分化研究。结果显示:(1)两个物种具有中等水平的遗传多样性;羌活的平均等位基因数(N_a)、有效等位基因数(N_e)和期望杂合度(H_e)分别为2.603、1.777和0.313,均高于宽叶羌活(分别为2.200、1.641和0.308)。(2)分子方差分析表明,两个物种的遗传变异主要存在于群体内,羌活和宽叶羌活群体间分化系数分别为0.181和0.191。(3)Structure聚类分析和主坐标分析(PCoA)将所有取样个体分为两大遗传组分,分别对应于羌活和宽叶羌活两个物种,二者间存在着有限的基因交流。研究表明,羌活与宽叶羌活物种间存在较高程度的遗传分化,并且遗传变异主要源自群体内,应各自划分为不同的地理单元进行多样性保护。