Pluripotent stem cells--model of embryonic development,tool for gene targeting,and basis of cell therapy

Embryonic stem (ES) cells are pluripotent cell lines with the capacity of self-renewal and a broad differentiation plasticity. They are derived from pre-implantation embryos and can be propagated as a homogeneous, uncommitted cell population for an almost unlimited period of time without losing their pluripotency and their stable karyotype. Murine ES cells are able to reintegrate fully into embryogenesis when returned into an early embryo, even after extensive genetic manipulation. In the resulting chimeric offspring produced by blastocyst injection or morula aggregation, ES cell descendants are represented among all cell types, including functional gametes. Therefore, mouse ES cells represent an important tool for genetic engineering, in particular via homologous recombination, to introduce gene knock-outs and other precise genomic modifications into the mouse germ line. Because of these properties ES cell technology is of high interest for other model organisms and for livestock species like cattle and pigs. However, in spite of tremendous research activities, no proven ES cells colonizing the germ line have yet been established for vertebrate species other than the mouse (Evans and Kaufman, 1981; Martin, 1981) and chicken (Pain et al., 1996). The in vitro differentiation capacity of ES cells provides unique opportunities for experimental analysis of gene regulation and function during cell commitment and differentiation in early embryogenesis. Recently, pluripotent stem cells were established from human embryos (Thomson et al., 1998) and early fetuses (Shamblott et al., 1998), opening new scenarios both for research in human developmental biology and for medical applications, i.e. cell replacement strategies. At about the same time, research activities focused on characteristics and differentiation potential of somatic stem cells, unravelling an unexpected plasticity of these cell types. Somatic stem cells are found in differentiated tissues and can renew themselves in addition to generating the specialized cell types of the tissue from which they originate. Additional to discoveries of somatic stem cells in tissues that were previously not thought to contain these kinds of cells, they also appear to be capable of developing into cell types of other tissues, but have a reduced differentiation potential as compared to embryo-derived stem cells. Therefore, somatic stem cells are referred to as multipotent rather than pluripotent. This review summarizes characteristics of pluripotent stem cells in the mouse and in selected livestock species, explains their use for genetic engineering and basic research on embryonic development, and evaluates their potential for cell therapy as compared to somatic stem cells.     

Protein displacement by DExH/D "RNA helicases" without duplex unwinding

Members of the DExH/D superfamily of nucleic acid-activated nucleotide triphosphatases are essential for virtually all aspects of RNA metabolism, including pre-messenger RNA splicing, RNA interference, translation, and nucleocytoplasmic trafficking. Physiological substrates for these enzymes are thought to be regions of double-stranded RNA, because several DExH/D proteins catalyze strand separation in vitro. These "RNA helicases" can also disrupt RNA-protein interactions, but it is unclear whether this activity is coupled to duplex unwinding. Here we demonstrate that two unrelated DExH/D proteins catalyze protein displacement independently of duplex unwinding. Therefore, the essential functions of DExH/D proteins are not confined to RNA duplexes but can be exerted on a wide range of ribonucleoprotein substrates.     

Assessment of the loose smut fungi (Ustilago nuda and U. tritici) in tissues of barley and wheat by fluorescence microscopy and real-time PCR

Loose smut fungi of barley and wheat (Ustilago nuda and U. tritici, respectively) colonize the plant without causing obvious disease symptoms before heading. The availability of diagnostic methods to detect and follow the growth of these pathogens in the plant would therefore be highly advantageous for both resistance breeding and the development of effective seed treatments. Using seed lots of barley and wheat highly infected with loose smut, we studied the early establishment of the loose smut pathogens in the plant by fluorescence microscopy. In hand-cut sections stained with the fluorochrome Blankophor?, fungal hyphae were observed to invade the shoot apical meristem and leaf primordia during the first days after the onset of germination. At the first node stage the ear and leaf primordia were generally extensively colonized. Hyphae of U. nuda were also regularly observed in high density in the nodes. A protocol was developed for the specific amplification of U. nuda and U. tritici DNA extracted from infected plant tissue. PCR screening of U nuda in seedlings from infected and healthy seed lots was compared to ELISA, microscopy and ultimately head infection of mature plants derived from tillers of the tested seedlings. The results indicated that a prediction of loose smut infection by real-time PCR is possible at the second leaf stage, and that the assay is equally suited for use with spring and winter varieties of barley and wheat.     

Structural and physiological changes associated with the skin spot disorder in apple

Skin spot is an important physiological disorder of ‘Elstar’ apples (Malus × domestica Borkh.) that occurs after fruit have been removed from controlled atmosphere storage. Skin spots are irregular patches of small, round, brown blemishes. Cross-sections reveal a browning of protoplasts (coagulated) and of cell walls that extends into the hypodermis. Skin spots are always associated with linear, gaping and non-gaping microcracks in the cuticle. Staining of apple skin with calcofluor white usually results in white fluorescence of cell walls but, within a skin spot, the white fluorescence is weak or absent. Cell walls within, and in the immediate vicinity of skin spots stain with phloroglucin/HCl indicating the presence of lignin. The area of skin affected by skin spots was positively and linearly correlated with the area of the non-blush fruit surface infiltrated by acridine orange. In general, skin spots were limited to the non-blush fruit surface and occurred more frequently near the stem-end than the calyx region of the fruit. Skin spot areas were correlated with a 2.5-fold increase in water vapour permeability compared with unaffected areas (23.8 ± 4.0 m s⁻¹ with skin spots, 9.6 ± 2.1 × 10⁻⁵ m s⁻¹ without skin spots). Strips of the fruit skin from regions with skin spots had an increased maximum force and modulus of elasticity. Dipping fruit in ascorbic acid (0.1 or 0.3 mM for 10 min) before storage decreased the area affected by skin spots. There was no effect of dipping in ethanol/water (70%, v/v, 15 min) or in solutions of captan (1.5 g L⁻¹, 10 min) or trifloxystrobin (0.1 g L⁻¹, 10 min). In contrast, prestorage treatment with 1-methylcyclopropene (630 nL L⁻¹ for 24 h) or poststorage incubation in H₂O₂ (10% for 2, 6, 10 and 16 h) increased skin spots. Our data are consistent with a typical cell response to an oxidative burst that seems to be focussed on particular regions of the ‘Elstar’ fruit surface by concentrations of cuticular microcracks, and that is possibly caused by reoxygenation injury upon removal from CA storage.     

Effects of nitrogen feeding for extraradical mycelium of Rhizophagus irregularis maize symbiosis incorporated with phosphorus availability

In terrestrial ecosystems, plants are frequently in symbiosis with arbuscular mycorrhizal fungi (AMF) with mineral nutrients and photosynthesis carbon exchanges in between. This research sought to identify the effects of phosphorus (P) levels on the nitrogen (N) uptake via extraradical mycelium (ERM) and the mycorrhizal growth response (MGR) of maize plants within the AMF symbiosis. Pots were separated into root compartments and hyphae compartments (HCs) with two layers of a 30‐μm mesh membrane and an air gap in between, where only hyphae could pass through, to avoid both N diffusion and root growth effects. Maize plants were inoculated with Rhizophagus irregularis with different N fertilization in HCs under two different P fertilization levels. Our results indicated that a strong increase in MGR with low‐P fertilization. The same tendency was not observed with high‐P fertilization, although both had a large increase in P concentration as a potential source of growth in shoot tissue of mycorrhizal plants. Substantial effects (10.5% more N) were observed in the case of high‐P availability for the host plants from ERM fed with N, whereas under low‐P conditions ERM may prioritize P uptake rather than N uptake. The AM fungi increase the uptake of N and P, which are most limiting in the soil with fewer forces from soil resources. In addition, there was still more P accumulated than N due to the high N for ERM with high‐P supply. Low N in HCs corresponded with a lower colonization rate in roots but with high hyphae density in HCs; this result suggest that N and P availability might change the ratio of extraradical to intraradical hyphae length.     

Contribution of methylated exudate flavonoids to the anti-inflammatory activity of Grindelia robusta

The flavonoid pattern of an acetonic extract of Grindelia robusta Nutt. was investigated in detail. The flavonoids were enriched by CC. In addition to twelve known methylated exudate flavonols four compounds were identified for the first time in G. robusta. Several substances of the flavonoid complex, among them the main compounds quercetin-3-methylether and 6-OH-kaempferol-3,6-dimethylether, were tested for their activity to inhibit neutrophil elastase. Quercetin-3-methylether was shown to be most active with an IC₅₀ of 19 µM, thus obviously contributing to the anti-inflammatory activity of the drug.     

Evaluation of non-chemical seed treatment methods for the control of Alternaria dauci and A. radicina on carrot seeds

The current study was initiated to evaluate the efficacy of physical methods (hot water, aerated steam, electron treatment) and agents of natural origin (resistance inducers, plant derived products, micro-organisms) as seed treatments of carrots for control of Alternaria dauci and A. radicina. Control of both Alternaria species by seed treatment with the resistance inducers was generally poor. Results were also not satisfactory with most of the formulated commercial micro-organism preparations. Based on the average of five field trials, one of these, BA 2552 (Pseudomonas chlororaphis), provided a low but significant increase in plant stand. Among the experimental micro-organisms, the best results were obtained with Pseudomonas sp. strain MF 416 and Clonostachys rosea strain IK726. A similar level of efficacy was provided by seed treatment with an emulsion (1%) of thyme oil in water. Good and consistent control was generally achieved with the physical methods aerated steam, hot water and electron treatment. Aerated steam treatment was, apart from the thiram-containing chemical standard, the best single treatment, and its performance may at least partially be due to extensive pre-testing, resulting in dosages optimally adapted to the respective seed lot. In some of the experiments the effect of the hot water treatment, which was tested at a fixed, not specifically adapted dosage, was significantly improved when combined with a Pseudomonas sp. MF 416 or C. rosea IK726 treatment. The results are discussed in relation to the outcome of experiments in which the same seed treatment methods and agents were tested in other seed-borne vegetable pathosystems.     

Control of seed-borne pathogens on legumes by microbial and other alternative seed treatments

Greenhouse trials were carried out in order to test the efficacy of different seed treatments as alternatives to chemicals against Colletotrichum lindemuthianum cause of anthracnose on bean and Ascochyta spp. cause of Ascochyta blights on pea, respectively. Resistance inducers, commercially formulated microorganisms, non-formulated selected strains of different microorganisms (fungi, bacteria and yeasts) and plant extracts were applied as dry or liquid seed treatments on naturally infested seeds. Seedling emergence and disease incidence and/or severity were recorded. Almost all seed treatments turned out to be ineffective in controlling the Ascochyta infections, which is in line with the literature stating that these pathogens are difficult to control. The only alternative treatments that gave some control of Ascochyta spp. were thyme oil and a strain of Clonostachys rosea. The resistance inducers tested successfully controlled infections of bean by C. lindemuthianum. Among the formulated microorganisms, Bacillus subtilis-based formulations provided the best protection from anthracnose. Some strains of Pseudomonas putida, a disease-suppressive, saprophytic strain of Fusarium oxysporum and the mustard powder-based product Tillecur also proved to be effective against bean anthracnose. However, among the resistance inducers as well as among the other groups, certain agents caused a significant reduction of plant emergence. Different alternative seed treatments can therefore be used for the control of C. lindemuthianum on bean, while on pea only thyme oil and a strain of Clonostachys rosea showed some effectiveness against Ascochyta spp.     

An octahedral coordination complex of iron(VI)

The hexavalent state, considered to be the highest oxidation level accessible for iron, has previously been found only in the tetrahedral ferrate dianion, FeO4(2-). We report the photochemical synthesis of another Fe(VI) compound, an octahedrally coordinated dication bearing a terminal nitrido ligand. M?ssbauer and x-ray absorption spectra, supported by density functional theory, are consistent with the octahedral structure having an FeN triple bond of 1.57 angstroms and a singlet d2(xy) ground electronic configuration. The compound is stable at 77 kelvin and yields a high-spin Fe(III) species upon warming.     

Analysis of diversity in populations of plant pathogens: the barley powdery mildew pathogen across Europe

Understanding population genetics and evolution within species requires recognition of variation within and between populations and the ability to distinguish between the potential causes of an observed distribution of variation. For this aim several established indices of diversity, and a novel one, were applied to population samples of the barley powdery mildew pathogen,Erysiphe graminis f. sp.hordei. Random spore samples were obtained from the air along transects through regions of interest across large parts of Europe in 1990. Significant geographical differences in diversity of virulence genotypes occurred among regional sub-samples. Diversity was highest in the samples from eastern Germany, Denmark and Austria, whereas the lowest values were found in the samples from Italy, southern France and parts of western Germany. Diversity in the pathogen population was generally related to the degree of diversification of host resistance in time and space, although there was considerable variation in ranking among different measures of diversity. Sensitivity to sample size proved to be the major problem with the use of several established indices of diversity. Working with very large sample sizes we used multiple random subsamples of various smaller sizes to determine how the mean index values changed with changing sample size. The Shannon index proved to be considerably affected by sample size, in contrast to the Simpson index that was therefore used as a global measure of diversity. Limits of confidence were estimated for the Simpson index using the bootstrap method of numerical resampling. The two aspects contributing to global diversity, richness and evenness, were considered separately to allow meaningful interpretation of the Simpson index. Random sub-sampling was used to reduce the influence of sample size differences for these measures. Dissimilarity, a novel measure of diversity for use in plant pathology, indicates the average number of major genes for host resistance against which pathotypes in a sample respond differently. It is thus able to account for the genetic relationship among pathotypes, which is not considered by any other index. The approaches developed in this study help to compare major forces driving evolution of large-scale populations of the barley mildew pathogen.