RNA silencing platforms in plants

Since the discovery of RNAi, its mechanism in plants and animals has been intensively studied, widely exploited as a research tool, and used for a number of potential commercial applications. In this article, we discuss the platforms for delivering RNAi in plants. We provide a brief background to these platforms and concentrate on discussing the more recent advances, comparing the RNAi technologies used in plants with those used in animals, and trying to predict the ways in which RNAi technologies may further develop.     

Determination of the specificity of monoclonal antibodies against Schistosoma mansoni CAA glycoprotein antigen using neoglycoconjugate variants

The immunogenic O-glycan of circulating anodic antigen (CAA) is a high-molecular-mass polysaccharide with the unique -->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GalpNAc-(1--> repeating unit. To obtain information at the molecular level about the specificity of monoclonal antibodies against CAA, the immunoreactivity of two series of bovine serum albumin-coupled synthetic oligosaccharides related to the CAA O-glycan was monitored using ELISA and surface plasmon resonance spectroscopy. The importance of the axial hydroxyl group of beta-D-GalpNAc for antibody binding was investigated using the following series of analogues: beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->O); beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O); and beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O). In the second series of analogues, beta-D-Glcp6S-(1-->3)-beta-D-GalpNAc-(1-->O), beta-D-GalpNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), and beta-D-Glcp6S-(1-->3)-beta-D-Gal-pNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), the native beta-D-GlcpA moiety was replaced by beta-D-Glcp6S to evaluate the influence of the nature of the charge on antibody recognition. Comparison of the immunoreactivity of these series with that measured for conjugates containing corresponding synthetic CAA fragments showed that the antibody binding levels can be correlated to the antibody specificity to CAA fragments. For the most reactive antibodies, the structural changes chosen (beta-D-GalpNAc replaced by beta-D-GlcpNAc, and beta-D-GlcpA replaced by beta-D-Glcp6S) completely eradicated the binding.     

Arabidopsis SENESCENCE-ASSOCIATED GENE101 stabilizes and signals within an ENHANCED DISEASE SUSCEPTIBILITY1 complex in plant innate immunity

Plant innate immunity against invasive biotrophic pathogens depends on the intracellular defense regulator ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). We show here that Arabidopsis thaliana EDS1 interacts in vivo with another protein, SENESCENCE-ASSOCIATED GENE101 (SAG101), discovered through a proteomic approach to identify new EDS1 pathway components. Together with PHYTOALEXIN-DEFICIENT4 (PAD4), a known EDS1 interactor, SAG101 contributes intrinsic and indispensable signaling activity to EDS1-dependent resistance. The combined activities of SAG101 and PAD4 are necessary for programmed cell death triggered by the Toll-Interleukin-1 Receptor type of nucleotide binding/leucine-rich repeat immune receptor in response to avirulent pathogen isolates and in restricting the growth of normally virulent pathogens. We further demonstrate by a combination of cell fractionation, coimmunoprecipitation, and fluorescence resonance energy transfer experiments the existence of an EDS1-SAG101 complex inside the nucleus that is molecularly and spatially distinct from EDS1-PAD4 associations in the nucleus and cytoplasm. By contrast, EDS1 homomeric interactions were detected in the cytoplasm but not inside the nucleus. These data, combined with evidence for coregulation between individual EDS1 complexes, suggest that dynamic interactions of EDS1 and its signaling partners in multiple cell compartments are important for plant defense signal relay.     

ATM regulates target switching to escalating doses of radiation in the intestines

Although stem cells succumbing to reproductive death are assumed to be the single relevant targets in radiation tissue damage, recent studies showed intestinal stem cell damage is conditionally linked to crypt endothelial apoptosis, defining a two-target model. Here we report that when mouse intestines were protected against microvascular apoptosis, radiation switched as the dose escalated to a previously unrecognized crypt stem cell target, activating ceramide synthase-mediated apoptosis to initiate intestinal damage. Whereas ataxia telangiectasia-mutated (ATM) kinase normally represses ceramide synthase, its derepression in Atm(-/-) mice increased crypt stem cell radiosensitivity 3.7-fold without sensitizing the microvascular response. Discovery of this intestinal radiosensitivity mechanism allowed design of an antisense Atm oligonucleotide treatment which phenocopied the Atm(-/-) mouse, reordering ceramide synthase-mediated stem cell death to become the first-line gastrointestinal response of wild-type littermates. These experiments indicate that tissues operate multiple potential targets activated consecutively according to their inherent radiosensitivities that may be reordered therapeutically to control radiation tissue responses.     

Fertility and hatchability of eggs laid in the pullet-to-breeder transition period and in the initial production period

The initial eggs produced by broiler breeder hens are relatively small compared with later in the production cycle. An evaluation of indices related to hatchability is required when these eggs are to be used for the production of broiler chicks. Two experiments were conducted to evaluate characteristics related to the hatchability of eggs from pullet-to-breeder transition phase, at 25 and 27 weeks of age, and from the peak of production period and five weeks later, at 32 and 37 weeks of age. Eggs from birds 25 weeks had a lesser fertility in Experiment 1. Mortality occurred unevenly in early (1–5 days), middle (6–17 days) and late (18–21 days) incubation, and greater mortality was observed after the internal membrane was ruptured. The younger the hen, the lighter the egg, chick, and shell, and the longer the time required to complete the hatching process. In Experiment 2, greater mortalities were observed at the early period (1–5 days) and after “pipping” of the internal and external membranes. Embryos from heavy eggs of breeder hens 37 weeks of age took less time to complete the hatching process. Results indicated the larger the egg, the heavier the chick and shell, and the lesser the shell percentage. As breeder age advanced, characteristics related to egg fertility and hatchability improved.     

Molecular mechanisms of action of angiopreventive anti-oxidants on endothelial cells: microarray gene expression analyses

The anti-oxidants N-acetyl-l-cysteine (NAC) and (-)-epigallocatechin-3-gallate (EGCG) inhibit tumor vascularization by reducing endothelial cell migration and invasion in a similar, non additive and non synergistic manner but do not alter the growth of human umbilical vein endothelial cells. Here we address the effects of the two chemopreventive drugs on endothelial cell signaling by means of expression profiling and real-time PCR validation. We identify a series of angiogenesis related genes that are similarly regulated by the two drugs. Anti-oxidant treated endothelial cells show gene expression profiles compatible with a less activated, less apoptosis prone and less migratory phenotype. The anti-oxidants affect expression of several components of the TNFalpha response pathway including downstream genes that are regulated in the opposite direction in the absence of the inflammatory cytokine. The interference with the TNFalpha pathway is reflected by reduced NFkappaB activation in anti-oxidants treated cells but the compounds are not able to contrast TNFalpha mediated activation of NFkappaB. The chemopreventive action of these compounds thus relies on a reduction of basal levels of endothelial cell activation. Down-regulation of the TNFalpha responsive pro-metastatic, pro-inflammatory genes, urokinase plasminogen activator and selectin E, further implies anti-metastatic effects for these drugs.     

Environmental genotoxicity assessment of an urban stream using freshwater planarians

Pollution is a major concern in urban areas. Due to its biological significance, genotoxicity should be a main focus for pollution biomonitoring, due mainly to the increasing complexity of the chemical environment in which organisms are exposed. Diluvio's Basin (Porto Alegre, RS, Brazil) is a heavily polluted urban ecosystem impacted by urban wastewater. Planarians are useful organism for evaluating environmental genotoxicity because of their high sensitivity, low cost, high proliferative rate and also because of their basal evolutionary position in relation to complex metazoans. Comet assay is a powerful and highly sensitive method of evaluating primary DNA lesions. Based on the unique features of planarians and the current environmental state of Diluvio's Basin, the aim of this work was to evaluate the genotoxic potential of this body of water using comet assay in planarians. Planarians were exposed to the water for 13 days in a laboratory and comet assay was performed in order to screen possible DNA damages. The results indicated an increasing gradient of damage towards basin's mouth. Such a gradient could be related to the gradual increase of pollutants among the different sample sites. Moreover, there seems to be a correlation between the urbanization gradient that exists within the watershed and the genotoxicity. Historical physical-chemical data was also gathered and examined for possible correlations with genotoxicity. Comet assay in planarians is a very promising test for environmental monitoring studies. Its application should be expanded.     

Insurance and other socioeconomic determinants of elderly longevity in a Costa Rican panel

Official figures show that life expectancy in Costa Rica is longer than in the United States (US), in spite of the fact that per capita health expenditure is only one-tenth that of the US. To check whether this is for real and to explore some of its determinants, 900 Costa Ricans aged 60+ were followed from 1984 to 2001. Follow-up household visits were made, deaths were tracked in the national death registry, and survival status in the voting registry was double-checked. In addition, the survivors were contacted in 2002. Two-thirds of the panel had died by December 2001. Kaplan-Meier curves, life tables and Cox regression were used to analyse the panel's survival. Mortality in the panel was slightly higher than the Costa Rican average and similar to that in the US, confirming the exceptional longevity of Costa Ricans. Survival was substantially lower among unmarried men and individuals with limited autonomy at the beginning of the study. The effect of socioeconomic status is weak. Insurance effects seem to be confounded by selection biases.     

Incubation of Metanephroi with Vitamin D3 Increases Numbers of Glomeruli

To characterize actions of vitamin D3 on metanephroi transplanted from rat embryos to adult recipients, we incubated metanephroi with or without 0.01, 0.1 or 1 ug/ml vitamin D3, 25-hydroxyvitamin D₃ [25(OH)D₃] or 1, 25-hydroxyvitamin D₃ [1,25(OH)2D₃] prior to implantation. The number of glomeruli in developed metanephroi three weeks post-transplantation that had been incubated with 1.0 ug/ml vitamin D₃ was increased relative to the number in metanephroi that were not incubated with vitamin D₃ (control), an effect that was not recapitulated by administration of vitamin D₃ directly to hosts at the time of transplantation. Incubation of metanephroi with 1.0 ug/ml vitamin D₃ also enhanced inulin clearances of metanephroi measured at 12 weeks post-transplantation. The hydroxylated derivative of vitamin D₃, 25(OH)D₃, increased glomerulus number when applied at 0.01 ug/ml but not at higher concentrations, while the twice-hydroxylated derivative 1,25(OH)₂D₃, failed to increase glomerulus number at any concentration tested. We conclude that incubation with vitamin D₃ prior to implantation enhances inulin clearance possibly by increasing the number of glomeruli that develop post-transplantation.Our findings suggest the vitamin D₃ effect is mediated locally.Key Words: kidney, organogenesis, transplantation