Transgenic rice plants: Characterization of two generations of seed progeny

Transgenic rice plants have been regenerated from kanamycin-resistant callus of Oryza sativa (cv. Taipei 309) derived from protoplasts electroporated with pCaMVNEO carrying the neomycin phosphotransferase II ( nptII ) gene. Of 6 randomly selected plants, all contained the nptll gene, but only 2 plants expressed NPTII activity. The transgenic plants were significantly shorter, produced fewer tillers, took longer to flower and had reduced fertility compared to non-transformed protoplastderived plants. Fifty-six seeds collected from one transgenic plant expressing NPTII activity germinated on medium containing kanamycin sulphate to give 16 green, first seed generation (R₁) plants. The latter could be divided into 3 groups: (i) Plants which set seed, had normal floret morphology and produced a total of 76 seeds; (ii) Plants which flowered, but which failed to set seed; (iii) Plants which failed to flower, were shorter and had significantly fewer tillers than plants of groups (i) and (ii). The nptII gene was present in all transgenic R₁ plants, but only 8 plants expressed the gene. Phenotypic characteristics, observed in transgenic R₁ plants were also seen in the transforned R₂ plants. These included reduced stature, a longer vegetative phase and reduced fertility compared to non-transformed plants.     

野生型rLj-RGD3基因重组突变体rLj-112分子克隆及对HeLa细胞活性影响

为研究突变体rLj-112蛋白的抗肿瘤活性,人工合成七鳃鳗野生型rLj-RGD3蛋白和突变型rLj-112蛋白,通过比较两种蛋白质的抗增殖、迁移和促凋亡的活性,确定突变体rLj-112蛋白的生物学意义及地位。采用MTT方法检测不同浓度的rLj-112蛋白对HeLa细胞增殖的抑制作用。结果表明,rLj-112蛋白能显著抑制HeLa细胞的增殖,且IC50为4.3 μmol/L。使用Transwell细胞培养板对bFGF诱导的HeLa细胞迁移实验表明,rLj-112蛋白能抑制HeLa细胞的迁移。rLj-112蛋白作用后,HeLa细胞经Hoechst33258和AnnexinV-FITC染色结果显示,细胞均凋亡。流式细胞仪进一步证明,rLj-112蛋白能诱导HeLa细胞发生凋亡,且呈剂量依赖性。由此可见,与野生型rLj-RGD3蛋白比较,突变型rLj-112蛋白有较高的细胞毒性作用,具有抗肿瘤的功能,有望应用于抗肿瘤基因工程药物的开发,具有重要的生物学意义。     

Adherens junctions and desmosomes are damaged by Entamoeba histolytica: Participation of EhCPADH complex and EhCP112 protease

Entamoeba histolytica trophozoites adhere to epithelium at the cell–cell contact and perturb tight junctions disturbing the transepithelial electrical resistance. Behind tight junctions are the adherens junctions (AJs) that reinforce them and the desmosomes (DSMs) that maintain the epithelium integrity. The damage produced to AJs and DMSs by this parasite is unknown. Here, we studied the effect of the trophozoites, the EhCPADH complex, and the EhCP112 recombinant enzyme (rEhCP112) on AJ and DSM proteins. We found that trophozoites degraded β‐cat, E‐cad, Dsp l/ll, and Dsg‐2 with the participation of EhCPADH and EhCP112. After contact of epithelial cells with trophozoites, immunofluorescence and transmission electron microscopy assays revealed EhCPADH and rEhCP112 at the intercellular space where they colocalised with β‐cat, E‐cad, Dsp l/ll, and Dsg‐2. Moreover, our results suggested that rEhCP112 could be internalised by caveolae and clathrin‐coated vesicles. Immunoprecipitation assays showed the interaction of EhCPADH with β‐cat and Dsp l/ll. Besides, in vivo assays demonstrated that rEhCP112 concentrates at the cellular borders of the mouse intestine degrading E‐cad and Dsp I/II. Our research gives the first clues on the trophozoite attack to AJs and DSMs and point out the role of the EhCPADH and EhCP112 in the multifactorial event of trophozoites virulence.     

rLj-RGD3全RGD缺失基因重组突变体——rLj-112的分子克隆及其广谱抑真菌活性

Lj-112是日本七鳃鳗RGD毒素蛋白野生型Lj-RGD3的RGD全缺失基因突变体,其一级结构具有富组氨酸的特点．富组氨酸糖蛋白具有抑菌功能．为了研究Lj-112是否具有抑菌作用,使用基因合成和原核表达的方法,获得了纯化重组蛋白rLj-112,将其对不同菌种进行抑菌试验.结果表明,以野生型rLj-RGD3、RGD模体与组氨酸全缺失突变体rLj-26为对照,rLj-112有较广谱的抑菌活性,其中对白念球菌的最低抑菌浓度（MIC）为7 μmol/L,对稻瘟病菌的MIC值为7.5 μmol/L,对毛癣菌和禾谷病菌的MIC值分别为11.9 μmol/L和29.9 μmol/L．可见,rLj-112能作为抗真菌药物候选,为提高农作物的生产质量和减轻人类感染疾病的用药压力奠定了基础．     

Positioning ribosomes on leader mRNA for translational activation of the message of an inducible Staphylococcus aureus cat gene

Summary The expression of the chloramphenicol (Cm) —inducible Cm acetyltransferase gene (cat) of the staphylococcal plasmid pUB112 is regulated at the translational level. The leader mRNA preceding the cat coding sequence can form a stable hairpin structure, in which the cat Shine-Dalgarno sequence is masked. Previous work showed that translation of a short leader peptide terminating within the stem of the inhibitory secondary structure is required for basal Cm acetyltransferase (CAT) synthesis and its inducibility. In the present study we shortened this leader peptide by introducing ochre codons in its coding sequence and found that synthesis of the N-terminal part of the leader peptide, terminating directly 5 to the stem, is sufficient to mediate basal and inducible CAT synthesis. Amino acid substitution in this region of the leader peptide abolished inducibility. We suggest that the 5 region of the leader peptide coding sequence specifies a particularly Cm-sensitive translation that represents the Cm-sensor mechanism for cat gene induction.Part of this work was presented at the Fourth International Conference on Genetics and Biotechnology of Bacilli, San Diego, USA, 1987 and at the Herbsttagung der Gesellschaft für Biologische Chemie, Erlangen, Federal Republic of Germany, 1987     

Photo-catalytic oxidation of a di-nuclear manganese centre in an engineered bacterioferritin ‘reaction centre’

Photosynthesis involves the conversion of light into chemical energy through a series of electron transfer reactions within membrane-bound pigment/protein complexes. The Photosystem II (PSII) complex in plants, algae and cyanobacteria catalyse the oxidation of water to molecular O₂. The complexity of PSII has thus far limited attempts to chemically replicate its function. Here we introduce a reverse engineering approach to build a simple, light-driven photo-catalyst based on the organization and function of the donor side of the PSII reaction centre. We have used bacterioferritin (BFR) (cytochrome b1) from Escherichia coli as the protein scaffold since it has several, inherently useful design features for engineering light-driven electron transport. Among these are: (i.) a di-iron binding site; (ii.) a potentially redox-active tyrosine residue; and (iii.) the ability to dimerise and form an inter-protein heme binding pocket within electron tunnelling distance of the di-iron binding site. Upon replacing the heme with the photoactive zinc-chlorin e₆ (ZnCe₆) molecule and the di-iron binding site with two manganese ions, we show that the two Mn ions bind as a weakly coupled di-nuclear Mn₂^II,II centre, and that ZnCe₆ binds in stoichiometric amounts of 1:2 with respect to the dimeric form of BFR. Upon illumination the bound ZnCe₆ initiates electron transfer, followed by oxidation of the di-nuclear Mn centre possibly via one of the inherent tyrosine residues in the vicinity of the Mn cluster. The light dependent loss of the Mn^II EPR signals and the formation of low field parallel mode Mn EPR signals are attributed to the formation of Mn^III species. The formation of the Mn^III is concomitant with consumption of oxygen. Our model is the first artificial reaction centre developed for the photo-catalytic oxidation of a di-metal site within a protein matrix which potentially mimics water oxidation centre (WOC) photo-assembly.     

Prevalence of Enterobius vermicularis Infection among Preschool Children in Kindergartens of Taipei City, Taiwan in 2008

The prevalence of Enterobius vermicularis infection among preschool children was reported to be low based on a 5-year screening program in Taipei City, Taiwan. The Taipei City government intended to terminate the E. vermicularis screening program among preschool children. Thus, we were entrusted with confirming whether pinworm infections among preschool children in Taipei City had truly declined. From each of 12 administrative districts 2-3 kindergartens were randomly selected for investigation. In total, 4,349 children were examined, of which 2,537 were boys and 1,812 were girls. The cellophane tape adhered to a glass slide was used, and all examinations were done by certified medical technologists. Results indicated that the overall prevalence rate of pinworm infections was 0.62% (27/4,349). Although the infection rate was higher among boys (0.67%, 17/2,537) than in girls (0.55%, 10/1,812), no significant difference was found (χ² = 0.399, P = 0.62). According to the administrative district, the infection rate ranged from no positive cases of E. vermicularis infection in the Xinyi, Zhongzhen, and Wanhua Districts (0%; 0/299, 0/165, and 0/358, respectively), to 0.26% (1/131) in Songshan District, with the highest rate of 1.88% (7/373) in Wenshan District. Because the overall infection rate (0.62%, 27/4,349) in the present study was unchanged compared to that (0.40%, 197/49,541) previously reported in 2005, we propose that regular pinworm screening and treatment programs should be continued in some parts of Taipei City.     

Symmetrically dividing cell specific division axes alteration observed in proteasome depleted C. elegans embryo

A fertilised Caenorhabditis elegans embryo shows an invariable pattern of cell division and forms a multicellular body where each cell locates to a defined position. Mitotic spindle orientation is determined by several preceding events including the migration of duplicated centrosomes on a nucleus and the rotation of nuclear-centrosome complex. Cell polarity is the dominant force driving nuclear-centrosome rotation and setting the mitotic spindle axis in parallel with the polarity axis during asymmetric cell division. It is reasonable that there is no nuclear-centrosome rotation in symmetrically dividing blastomeres, but the mechanism(s) which suppress rotation in these cells have been proposed because the rotations occur in some polarity defect embryos. Here we show the nuclear-centrosome rotation can be induced by depletion of RPN-2, a regulatory subunit of the proteasome. In these embryos, cell polarity is established normally and both asymmetrically and symmetrically dividing cells are generated through asymmetric cell divisions. The nuclear-centrosome rotations occurred normally in the asymmetrically dividing cell lineage, but also induced in symmetrically dividing daughter cells. Interestingly, we identified RPN-2 as a binding protein of PKC-3, one of critical elements for establishing cell polarity during early asymmetric cell divisions. In addition to asymmetrically dividing cells, PKC-3 is also expressed in symmetrically dividing cells and a role to suppress nuclear-centrosome rotation has been anticipated. Our data suggest that the expression of RPN-2 is involved in the mechanism to suppress nuclear-centrosome rotation in symmetrically dividing cells and it may work in cooperation with PKC-3.     

Variations in the morphology of rice plants regenerated from protoplasts using different culture procedures

Rice protoplasts were cultured using 4 different culture procedures such as agarose embedding (AE) without feeder cells and the use of filter membranes (MEM), one layer of nylon mesh (MS1), or a double layer of nylon mesh (MS2) with the inclusion of Lolium multiflorum as feeder cells. The protoplast plating efficiency was highest on the MEM, followed by MS2, MS1 and AE. However, plant regeneration frequencies were highest for MS1, followed by MS2, MEM and AE. The protoclonal plants differed in the morphology of leaves, flowers, spikelets, and panicles in comparison to seed-derived plants. They varied in almost every phenotypic characters evaluated. In many cases, the variation was significantly different in characteristics such as plant height, flag leaf length and width and ratio, and in panicle characteristics such as panicle length, number of primary branches, and number of spikelets per panicle. The number of seeds per panicle was greatly reduced in protoclonal plants when compared with seed-derived control plants. The seeds showed also significant differences in grain length and width in comparison to the control plants. Among the 4 groups of protoclonal plants derived from the 4 different culturing procedures themselves, there were also variations in almost all the phenotypic characteristics assessed. This revised version was published online in June 2006 with corrections to the Cover Date.     

山西口岸全国首次截获危险性蚧虫并蛎蚧

【目的】并蛎蚧属盾蚧科并蛎蚧属,该虫食性杂,危害植物种类多,极易随水果、苗木、景观花卉植物等寄主传播入境。并蛎蚧分布于美国、日本和我国台湾省,在我国大陆地区尚未见该虫的发生报道。2016年5月山西口岸从来自台北的水果释迦上截获了并蛎蚧,为我国大陆首次截获。【方法】通过显微形态特征的观察和测量进行种类鉴定,并研究并蛎蚧的生物学、生态学相关资料,对其进行风险评估。【结果】并蛎蚧与近似种的主要区别:中臀叶与第二臀叶愈合中间有腺刺,有第三对臀叶,中臀叶有向内延伸的硬化区。通过风险评估认为,来自我国台湾、日本的并蛎蚧入侵我国大陆的风险较大。并蛎蚧在大陆亚热带地区的定殖和扩散风险很大。【结论】我国大陆地区的气候条件、植物种类可为并蛎蚧的定殖提供可能的适生条件,各口岸应对进境的并蛎蚧寄主植物加强检验检疫。