Ri-plasmid as a helper for introducing vector DNA into alfalfa plants

Genetic engineering of legumes and other important dicotyledonous plants is limited because of the difficulty of regenerating plants via cell culture. Since a considerable number of crop plants can be regenerated only from root culture, the introduction of foreign genes into Agrobacterium rhizogenes-induced hairy roots may expand the list of crop plants that could be genetically engineered. Here we report genetic transformation of alfalfa (Medicago sativa L.), a valuable forage legume, using a virulent strain of Agrobacterium rhizogenes containing, in addition to its Ri-plasmid, a binary vector containing a nopaline synthase gene. Plant cells transformed by this vector can be easily identified by their ability to produce nopaline. Transformed alfalfa plants were recovered from A. rhizogenes-induced hairy roots. These transgenic plants were characterized by normal leaf morphology and stem growth but a root system that was shallow and more extensive than normal. These plants were also fertile, set seeds upon self-pollination and outcrossing. Nopaline was detected in R1 progeny. Southern blot analysis confirmed the presence of multiple copies of T-DNAs from the Riplasmid in the plant genome in addition to the vector T-DNA.     

Formation of novel bioactive metabolites from the reactions of pro-inflammatory oxidants with polyphenolics

Dietary polyphenolics such as those in soy or red wine can have beneficial effects on the development of chronic human diseases. The mechanisms of action of isoflavones have been diverse and include their roles as weak estrogens, inhibitors of tyrosine kinase-dependent signal transduction processes and as antioxidants. Recent insights into the oxidative stress model of atherosclerosis suggest an interesting synthesis of these concepts. Sites of inflammation are associated with the formation of complex mixtures of reactive oxygen, nitrogen and halogenating species capable of modifying both endogenous biomolecules and polyphenolics. Of particular significance are the halogenation reactions mediated by myeloperoxidase that can modify key amino acids such as arginine and polyphenolics such as genistein. Hypochlorite, the reaction product of myeloperoxidase can halogenate polyphenolics to form stable derivatives with modified biological activity. Thus the in situ metabolism at sites of inflammation is unique and generates novel pharmacophores with potentially distinct modes of action from the parent compounds.     

A case of promiscuity: Agrobacterium's endless hunt for new partners

Agrobacterium tumefaciens is a phytopathogenic bacterium that induces the 'crown gall' disease in plants by transfer and integration of a segment of its tumor-inducing (Ti) plasmid DNA into the genome of numerous plant species that represent most of the higher plant families. Recently, it has been shown that, under laboratory conditions, the host range of Agrobacterium can be extended to non-plant eukaryotic organisms. These include yeast, filamentous fungi, cultivated mushrooms and human cultured cells. In this article, we present Agrobacterium-mediated transformation of non-plant organisms as a source of new protocols for genetic transformation, as a unique tool for genomic studies (insertional mutagenesis or targeted DNA integration) and as a useful model system to study bacterium-host cell interactions. Moreover, better knowledge of the DNA-transfer mechanisms from bacteria to eukaryotic organisms can also help in understanding horizontal gene transfer--a driving force throughout biological evolution.     

Polyphenolics from peach (Prunus persica var. Rich Lady) inhibit tumor growth and metastasis of MDA-MB-435 breast cancer cells in vivo

The tumor growth inhibition and anti-metastatic effects of peach polyphenolics were investigated in vivo using a xenograft model and MDA-MB-435 breast cancer cells. Results showed that tumor growth and lung metastasis were inhibited in vivo by peach polyphenolics in a dose range of 0.8–1.6 mg/day, and these effects were mediated by inhibition of metalloproteinases gene expression. Modulation of metalloproteinase-2, metalloproteinase-3 and metalloproteinase-13 gene expression may be some of the molecular targets for anti-metastatic activity of peach polyphenolics. Therefore, these compounds may constitute a novel chemopreventive tool to reduce the risk of metastasis in the combination therapy when primary cancer is diagnosed. Conversion to equivalent human intake for future clinical studies using the body surface area normalization method gave a dose of ~370.6 mg/day for a human adult of 60 kg, which can be supplied by consuming two to three peach fruit per day or alternatively using a dietary supplement peach polyphenol extract powder.     

Applied environmental stresses to enhance the levels of polyphenolics in leaves of hawthorn plants

In this investigation, two species of Crataegus (hawthorn) were chosen because their polyphenolic constituents have recently received greater attention for the treatment of patients with severe heart disease. One-year-old plants of hawthorn ( Crataegus laevigata and C. monogyna ) were subjected to water-deficit (continuous water deprivation), cold (4°C), flooding (immersion of roots of plants in water) or herbivory (leaf removal) stress treatments (each of 10 days duration) in order to assess their effects on levels of polyphenolics, namely (-)-epicatechin, catechin, chlorogenic acid, vitexin, vitexin-2'- O -rhamnoside, acetylvitexin-2'- O -rhamnoside, hyperoside, quercetin, and rutin in the leaves. The working hypothesis followed is that one or more of these stress treatment will elicit increases in the levels of these polyphenolics. Cold stress causes increases in levels of vitexin-2'- O -rhamnoside, acetylvitexin-2'- O -rhamnoside, hyperoside, and quercetin in both Crataegus species. Water-deficit stress increased the productivity of chlorogenic acid, catechin, and (-)-epicatechin in both hawthorn species. Flooding and herbivory caused no net increases, and in some cases, decreases in levels of polyphenolics. These studies indicate that either water-deficit stress or cold stress treatments, or a combination of the two, can be used to enhance the levels of desired polyphenolics in the leaves of these two hawthorn species in a photobioreactor system. These results may have significance for hawthorn in adapting to water-deficit or cold stress and are important considerations for the use of hawthorn in the treatment of heart disease in humans.     

Loss of heterocyclic amine mutagens by insoluble hemicellulose fiber and high-molecular-weight soluble polyphenolics of coffee

The presence of 2 kinds of components in brewed and instant coffee that could remove and destroy heterocyclic amine mutagens was demonstrated. The component that could remove the mutagens was insoluble fiber composed of hemicellulose. The fiber could tightly adsorb the mutagens Trp-P-1, Trp-P-2, Glu-P-1 and A alpha C, and those generated in roasted coffee beans. The component that could destroy the mutagens was high-molecular-weight soluble polyphenolics. They might be converted into quinone derivatives in the presence of molecular oxygen. The quinone derivatives might destroy the mutagens. The fibers and the polyphenolics in one cup of brewed or instant coffee had the capacity to remove and destroy a substantial amount of the mutagens in pyrolysates of foodstuffs.     

Induction of the alkA gene of Escherichia coli in gram-negative bacteria.

A broad-host-range plasmid containing a fusion of the alkA and lacZ genes of Escherichia coli was introduced into various aerobic and facultative gram-negative bacteria--33 species belonging to 19 genera--to study the induction of expression of the alkA gene by alkylating agents. The bacteria included species of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Vibrionaceae, Neisseriaceae, Rhodospirillaceae, and Azotobacteraceae. Results obtained show that all bacteria tested, except Aeromonas hydrophila, Agrobacterium tumefaciens, Hafnia alvei, Rhizobium meliloti, Salmonella enteritidis, Xanthomonas campestris, and those of the genus Rhodobacter, are able to induce the alkA gene of E. coli in the presence of N-methyl-N'-nitro-N-nitrosoguanidine. All these data indicate that the adaptive response to alkylating agents is present in bacterial species of several families and that the Ada box sequence must be widely conserved.     

Linkage and LOH studies in 19 cylindromatosis families show no evidence of genetic heterogeneity and refine the CYLD locus on chromosome 16q12–q13

Familial cylindromatosis is an autosomal dominant predisposition to multiple neoplasms of the skin appendages. The susceptibility gene has previously been mapped to chromosome 16q12-q13 and has features of a recessive oncogene/tumour suppressor gene. We have now evaluated 19 families with this disease by a combination of genetic linkage analysis and loss of heterozygosity in cylindromas from affected individuals. All 15 informative families show linkage to this locus, providing no evidence for genetic heterogeneity. Recombinant mapping has placed the gene in an interval of approximately 1 Mb. There is no evidence, between families, of haplotype sharing that might be indicative of common founder mutations.     

A preliminary molecular phylogenetic assessment of the lichen moths (Lepidoptera: Erebidae: Arctiinae: Lithosiini) with comments on palatability and chemical sequestration

The lichen moth tribe Lithosiini is best known for its hypothesized larval feeding behaviour, lichenivory. The larvae of some species have been found to be capable of sequestering polyphenolics, and the adults of some species are unpalatable to vertebrate predators. However, the chemical basis for the defence is unknown. Here we reconstruct a phylogenetic hypothesis using likelihood methods (maximum likelihood, Bayesian inference) for 65 species of Lithosiini representing 37 genera and seven outgroup species using 2806 bp of sequence data obtained from two mitochondrial gene fragments (COI barcoding region, CytB), and two nuclear gene fragments (RpS5 and ribosomal gene region 28S). Lithosiine species representing four of the seven subtribes and unplaced taxa are included in the analysis. The deeper relationships within the tribe are not strongly supported, and the monophyly of three of the four subtribes is not supported. The placement of the fourth subtribe, Acsalina, is weakly supported. We also conduct a survey of the secondary metabolites present within adults of five species. The species are examined for the presence of lichen polyphenolics and plant secondary metabolites sequestered by arctiines. Seven lichen polyphenolics are identified in the taxa examined, but no plant secondary metabolites are found using either analytical method. The results of this study and prior reports in the literature of chemical sequestration and unpalatability are plotted onto the resulting Bayesian inference phylogeny to examine the evolution of chemical defence within Lithosiini. Species that sequester lichen polyphenolics and unpalatable species occur in each of the major clades recovered in the analysis.     

PES1与雌激素受体存在相互作用

雌激素(E2)和雌激素受体(ER)在E2诱发的肿瘤中起着极其重要的作用.ER共调节因子通过与ER相互作用调节其生物学功能.PES1主要表达于E2的重要靶器官如乳腺、卵巢等组织中,并在乳腺癌细胞中高表达.用PCR技术构建HA标签的PES1全长以及1～322aa、312 ～588aa和414～588aa三个不同功能区片段的重组质粒.将不同的重组质粒与FLAG-ERα和或FLAGC-ERβ共转染293T细胞后进行免疫共沉淀,以验证PES1与ER是否有相互作用以及相互作用的区域.用含雌激素受体作用元件的荧光素酶报告基因( ERE-LUC)检测PES1对ERα和ERβ转录激活活性的影响.结果表明,PES1与ERα和ERβ均相互作用,且PES1的1～ 322aa区域与ERα和ERβ相结合.PES1能特异地、E2非依赖性抑制ERβ的转录激活活性.实验结果显示,PES1是一个新的ER共调节因子,需要进一步研究其在ERβ信号通路及其在E2诱发的肿瘤的作用.     

BROWN ALGAL POLYPHENOLS: PRIMARY METABOLITES WITH MULTIPLE,TRANSITIONAL ROLES

Arnold, Thomas M. Department of Biology, College/University of Charleston, Charleston S.C. 29424 Brown algal polyphenolics serve multiple functions. As defensive metabolites, they may serve to deter grazers, inhibit microbes, or absorb harmful UV radiation. However, these compounds are not classic ‘secondary metabolites’; they also have primary functions as cell wall strengtheners, spermatozoid inhibitors, and putative elements of algal holdfasts. These civilian roles of algal polyphenolics, which have been largely ignored in studies of plant defense, have implications for estimates of polyphenol cost. For example, in studies where growth-defense tradeoffs are used to indicate a cost for a multi-functional metabolite, the true cost is likely to have been significantly overestimated. To illustrate this, I propose a mechanism by which apparent growth vs. defense trade-offs can be predicted in the absence of a true cost of algal polyphenolics or in the absence of any defensive role for these compounds. This transitional-role model is based on the progression, or “aging” of phenolics and differentiates between physode-bound (reactive) polyphenols and the oxidized (unreactive) wall components. It predicts that variations in the levels of brown algal phenolics measured by colorimetry result from changes in the synthesis –secretion balance. Potential tests of this model, including pathway- and gene-level assessments of polyphenolic metabolism in brown algae, will be presented.     

Agroinfiltration is a versatile tool that facilitates comparative analyses of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis

The avirulence genes Avr9 and Avr4 from the fungal tomato pathogen Cladosporium fulvum encode extracellular proteins that elicit a hypersensitive response when injected into leaves of tomato plants carrying the matching resistance genes, Cf-9 and Cf-4, respectively. We successfully expressed both Avr9 and Avr4 genes in tobacco with the Agrobacterium tumefaciens transient transformation assay (agroinfiltration). In addition, we expressed the matching resistance genes, Cf-9 and Cf-4, through agroinfiltration. By combining transient Cf gene expression with either transgenic plants expressing one of the gene partners, Potato virus X (PVX)-mediated Avr gene expression, or elicitor injections, we demonstrated that agroinfiltration is a reliable and versatile tool to study Avr/Cf-mediated recognition. Significantly, agroinfiltration can be used to quantify and compare Avr/Cf-induced responses. Comparison of different Avr/Cf-interactions within one tobacco leaf showed that Avr9/Cf-9-induced necrosis developed slower than necrosis induced by Avr4/Cf-4. Quantitative analysis demonstrated that this temporal difference was due to a difference in Avr gene activities. Transient expression of matching Avr/Cf gene pairs in a number of plant families indicated that the signal transduction pathway required for Avr/Cf-induced responses is conserved within solanaceous species. Most non-solanaceous species did not develop specific Avr/Cf-induced responses. However, co-expression of the Avr4/Cf-4 gene pair in lettuce resulted in necrosis, providing the first proof that a resistance (R) gene can function in a different plant family.     

Regeneration of transgenic loblolly pine (Pinus taeda L.) from zygotic embryos transformed with Agrobacterium tumefaciens

Embryos of 24 open-pollinated families of loblolly pine (Pinus teade L.) were used as explants to conduct in vitro regeneration. Then, Agrobacterium tumefaciens strain GV3101 harboring the plasmid pPCV6NFHygGUSINT was used to transform mature zygotic embryos of seven families of loblolly pine. The frequency of transformation varied among families infected with A. tumefaciens. The highest frequency (100%) of transient beta-glucuronidase (GUS)-expressing embryos was obtained from family 11-1029 with over 300 blue spots per embryo. Expression of the GUS reporter gene was observed in cotyledons, hypocotyls, and radicles of co-cultivated mature zygotic embryos, as well as in callus and shoots derived from co-cultivated mature zygotic embryos. Ninety transgenic plants were regenerated from hygromycin-resistant callus derived from families W03. 8-1082 and 11-1029. and 19 transgenic plantlets were established in soil. The presence of the GUS gene in the plant genome was confirmed by polymerase chain reaction. Southern blot, and plant DNA/T-DNA junction analysis. These results suggest that an efficient A. tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for future studies on transferring economically important genes to loblolly pine.     

Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism.

Mutants of Agrobacterium tumefaciens which affect virulence or the ability to catabolize octopine were isolated after Tn5-induced mutagenesis. Of 8,900 colonies tested, 7 mutants with Tn5 insertions in a specific region of other Ti plasmid unable to catabolize octopine were isolated. Thirty-seven mutants affected in tumorigenesis resulted from insertions in the Ti plasmid and the Agrobacterium chromosome. Of these mutations, 12 were chromosomal and 25 mapped on the plasmid. Twenty-three mapped within a 20-megadalton region, which is distinct from the Ti plasmid sequences found stably integrated into the plant cell genome T-deoxyribonucleic acid). Included in these were mutants that were either a virulent or produced tumors with unusual morphologies. Three mutants contained insertions in the T-deoxyribonucleic acid. These three mutants incited tumors which synthesized octopine but had an altered morphology due to either extensive proliferation of shoots or roots from the tumor callus. Three additional mutants not caused by Tn5 contained mutations in the Ti plasmid.     

Red wine polyphenolics suppress the secretion and the synthesis of Apo B48 from human intestinal CaCo-2 cells

Epidemiological studies suggest that the red wine consumption may reduce the risk factor of cardiovascular disease. However, the mechanisms of how the red wine phenolic components reduce the risk of cardiovascular disease is currently unknown. Our previous study demonstrated that red wine polyphenolics suppress the secretion of pro-atherogenic lipoproteins (very low density lipoproteins) from human hepatic HepG2 cells. Therefore, in this study we hypothesize that red wine polyphenolics will also attenuate the production and secretion of another pro-atherogenic lipoprotein (chylomicrons) from human intestinal CaCo-2 cells. Cultured CaCo-2 cells were incubated in the presence of dealcoholized red wine, alcoholized red wine and atorvastatin for 24 h. The apo B48 protein (marker of intestinal chylomicrons) was quantified on Western blotting and the enhanced chemiluminescence. Apo B48 levels in the cells and that secreted into the media were significantly reduced by 29% in the cells incubated with dealcoholized red wine compared with control cells. Also the similar effect was shown in the cells incubated with alcoholized red wine. The cells incubated with atorvastatin shown the significant reduction of apo B48 production compared to control cells. Collectively, this study suggests that red wine polyphenolics down regulate the production of chylomicron in intestinal CaCo-2 cells.     

Complementation analysis of Agrobacterium tumefaciens Ti plasmid mutations affecting oncogenicity

A wide host range cosmid vector has been constructed by insertion of the lambda cos site into the plasmid pRK2501. This cosmid, which is maintained in Agrobacterium tumefaciens and is compatible with the Ti plasmid, has been used to make a clone bank of the A. tumefaciens pTiA6 plasmid. Several pTiA6 cosmids have been used to complement Tn5-induced Ti plasmid mutations. Five avirulent mutations which map outside of the region of the plasmid maintained in plant tumours (T-DNA) could be complemented in a trans orientation. Two mutations which are located on a single HpaI restriction fragment outside of the T-DNA, as well as three mutations which map within the T-DNA region, could not be complemented in a trans orientation in a REC- host.     

The Susceptibility of Monocotyledons to Agrobacterium tumefaciens

The susceptibility of 257 monocotyledon species belonging to 139 genera and 27 families, has been tested and the literature on this subject reviewed. In contrast with dicotyledons and gymnosperms, monocotyledons are much less susceptible to Agrobacterium tumefaciens: only 3 % of the species of monocotyledons tested were host plants, whereas 60 % of the dicotyledons and gymnosperms were susceptible. Only the closely related monocotyledon orders Liliales (6 families) and Arales (1 family) contained species that were susceptible. These results are consistent with a proposed taxonomic relationship between the Liliales and the dicotyledons. A correlation between host biochemical capabilities and susceptibility to crown gall is suggested.     

Radical chemistry of epigallocatechin gallate and its relevance to protein damage

The radical chemistry of the plant polyphenolics epigallocatechin gallate (EGCG) and epigallocatechin (EGC) were investigated using electron paramagnetic resonance spectroscopy. Radical species formed spontaneously in aqueous solutions at low pH without external oxidant and were spin stabilized with Zn(II). The spectra were assigned to the gallyl radical and the anion gallyl radical, with only 10% of the signal assigned to a radical from the galloyl ester. Spectral simulations were used to establish a pK(a) of 4.8 for the EGCG radical and a pK(a) of 4.4 for the EGC radical. The electrochemical redox potentials of EGCG and EGC varied from 1000 mV at pH 3 to 400 mV at pH 8. The polyphenolics did not produce hydroxyl radicals unless reduced metal ions such as iron(II) were added to the system. Zinc(II)-stabilized EGCG radicals were more effective protein-precipitating agents than unoxidized EGCG and produced irreversibly complexed protein. EGCG and other naturally occurring polyphenolics are effective radical scavengers but their radical products have the potential to damage biological molecules such as proteins.     

离子注入水稻愈伤组织提高农杆菌转化效率的初步研究

通过ＧＵＳ报告基因表达的组织化学染色分析,我们发现低能氮离子束注入水稻愈伤组织可显著提高根癌农杆菌介转基因效率,这为克服单子叶植物对根癌农杆菌不敏感性、提高遗传转化效率的研究提供了一条新的途径。