MODULATION OF OSTEOGENIC DIFFERENTIATION IN HUMAN SKELETAL CELLS IN VITRO BY 5-AZACYTIDINE

Cellular differentiation is controlled by a variety of factors including gene methylation, which represses particular genes as cell fate is determined. The incorporation of 5-azacytidine (5azaC) into DNA in vitro prevents methylation and thus can alter cellular differentiation pathways. Human bone marrow fibroblasts and MG63 cells treated with 5azaC were used as models of osteogenic progenitors and of a more mature osteoblast phenotype, respectively. The capacity for differentiation of these cells following treatment with glucocorticoids was investigated. 5azaC treatment led to significant expression of the osteoblastic marker alkaline phosphatase in MG63 osteosarcoma cells, which was further augmented by glucocorticoids; however, in human marrow fibroblasts alkaline phosphatase activity was only observed in glucocorticoid-treated cultures. MG63 cells represent a phenotype late in the osteogenic lineage in which demethylation is sufficient to induce alkaline phosphatase activity. Marrow fibroblasts are at an earlier stage of differentiation and require stimulation with glucocorticoids. In contrast, the expression of osteocalcin, an osteoblastic marker, was unaffected by 5azaC treatment, suggesting that regulation of expression of the osteocalcin gene does not involve methylation. These models provide novel approaches to the study of the control of differentiation in the marrow fibroblastic system.     

Environmental change and the phenology of European aphids

Aphids, because of their short generation time and low developmental threshold temperatures, are an insect group expected to respond particularly strongly to environmental changes. Forty years of standardized, daily data on the abundance of flying aphids have been brought together from countries throughout Europe, through the EU Thematic Network 'EXAMINE'. Relationships between phenology, represented by date of first appearance in a year in a suction trap, of 29 aphid species and environmental data have been quantified using the residual maximum likelihood (REML) methodology. These relationships have been used with climate change scenario data to suggest plausible changes in aphid phenology. In general, the date of first record of aphid species in suction traps is expected to advance, the rate of advance varying with location and species, but averaging 8 days over the next 50 years. Strong relationships between aphid phenology and environmental variables have been found for many species, but they are notably weaker in species living all year on trees. Canonical variate analysis and principal coordinate analysis were used to determine ordinations of the 29 species on the basis of the presence/absence of explanatory variables in the REML models. There was strong discrimination between species with different life cycle strategies and between species feeding on herbs and trees, suggesting the possible value of trait-based groupings in predicting responses to environmental changes.     

Predicting impacts of climate-induced range expansion: an experimental framework and a test involving key grazers on temperate rocky shores

Climate change has strong potential to modify the structure and functioning of ecosystems, but experimental field studies into its effects are rare. On rocky shores, grazing limpets strongly affect ecosystem structure and their distribution in NW Europe is changing in response to climate change. Three limpet species co-occur in SW Britain ( Patella vulgata, Patella ulyssiponensis and Patella depressa ) on open rock and in pools. Shores in Ireland are similar, but currently lack P. depressa . It is anticipated that P. depressa will expand its range into Ireland as the climate warms, but we currently lack an empirical basis to predict the consequences of this change. Recent studies show that increasing abundance of P. depressa on British shores has been accompanied by a decline of P. vulgata suggesting interspecific competition. In this study, a new experimental framework was used to examine the potential for P. depressa to affect P. vulgata on Irish shores. P. vulgata was experimentally transplanted into enclosures on open rock and in pools in both Ireland and Britain. In pools, treatments also included transplanted P. ulyssiponensis to mimic natural assemblages. Growth and mortality of P. vulgata were measured over 6 months with no differences between Ireland and Britain. In Britain, P. vulgata caged in pools with transplanted P. depressa and P. ulyssiponensis showed reduced growth, compared with when caged in pools with P. ulyssiponensis alone. There was no effect of P. depressa on the growth rate of P. vulgata on open rock. Results indicate that if the range of P. depressa extends into Ireland, it would reduce the growth of P. vulgata where it co-occurs with P. ulyssiponensis in pools. The framework used here provides a field-based approach that could be used to examine the impacts of climate-induced range expansions on the structure and functioning of other ecosystems.     

Gametogenic cycles and reproduction in the beadlet sea anemone Actinia equina (Cnidaria: Anthozoa)

Sexual Actinia equina have been maintained in the laboratory. Monthly biopsies show that these animals have annual gametogenic cycles. Regular samples from several natural populations suggest that animals in the wild show similar gametogenic periodicity. In the laboratory, males release sperm, but females brood young, at least for a few weeks. Members of these broods are released sporadically, usually after a water change. Males and non-sexual adults kept in isolation for over a year release a few juveniles during and at the end of that time. These young are likely to be asexually reproduced. There appears to be geographical variation of anemone size and in the frequency of sexual individuals in populations round the British Coast, so that modes of reproduction may vary.     

Eleven polymorphic microsatellite markers for paternity analysis in the pectoral sandpiper,Calidris melanotos

Eleven polymorphic microsatellite markers were isolated for the pectoral sandpiper, Calidris melanotos. The number of alleles observed in a sample of 149 presumably unrelated adults ranged from nine to 23 with an observed heterozygosity ranging from 0.79 to 0.92. The set of markers described here will prove useful for accurately determining paternity and therefore elucidate the hitherto unknown mating system of this species. We also report on cross‐species amplification of these markers in the semipalmated sandpiper, Calidris pusilla.     

Factors Controlling the Abundance and Size Distribution of the Phototrophic Ciliate Myrionecta rubra in Open Waters of the North Atlantic

ABSTRACT. Myrionecta rubra, a ubiquitous planktonic ciliate, has received much attention due to its wide distribution, occurrence as a red tide organism, and unusual cryptophyte endosymbiont. Although well studied in coastal waters, M. rubra is poorly examined in the open ocean. In the Irminger Basin, North Atlantic, the abundance of M. rubra was 0–5 cells/ml, which is low compared with that found in coastal areas. Distinct patchiness (100 km) was revealed by geostatistical analysis. Multiple regression indicated there was little relationship between M. rubra abundance and a number of environmental factors, with the exception of temperature and phytoplankton biomass, which influenced abundance in the spring. We also improve on studies that indicate distinct size classes of M. rubra; we statistically recognise four significantly distinct width classes (5–16, 12–23, 18–27, 21–33 μm), which decrease in abundance with increasing size. A multinomial logistic regression revealed the main variable correlated with this size distribution was ambient nitrate concentration. Finally, we propose a hypothesis for the distribution of sizes, involving nutrients, feeding, and dividing of the endosymbiont.     

Use of gaseous 13NH3 administered to intact leaves of Nicotiana tabacum to study changes in nitrogen utilization during defence induction

Nitrogen‐13 (t_1/2 9.97 m), a radioactive isotope of nitrogen, offers unique opportunities to explore plant nitrogen utilization over short time periods. Here we describe a method for administering ¹³N as gaseous ¹³NH₃ to intact leaves of Nicotiana tabacum L. (cv Samsun), and measuring the labelled amino acids using radio high‐performance liquid chromatography (HPLC) on tissue extract. We used this method to study the effects of defence induction on plant nitrogen utilization by applying treatments of methyl jasmonate (MeJA), a potent defence elicitor. MeJA caused a significant increase relative to controls in key [¹³N]amino acids, including serine, glycine and alanine by 4 h post‐treatment, yet had no effect on ¹³NH₃ incorporation, a process that is primarily under the control of the glutamine synthatase/glutamate synthase pathway (GS/GOGAT) in cellular photorespiration. We suggest that the reconfiguration of nitrogen metabolism may reflect induction of non‐photorespiratory sources of nitrogen to better serve the plant's defences.     

Extracellular superoxide production, viability and redox poise in response to desiccation in recalcitrant Castanea sativa seeds

Reactive oxygen species (ROS) are implicated in seed death following dehydration in desiccation-intolerant 'recalcitrant' seeds. However, it is unknown if and how ROS are produced in the apoplast and if they play a role in stress signalling during desiccation. We studied intracellular damage and extracellular superoxide (O₂^·−) production upon desiccation in Castanea sativa seeds, mechanisms of O₂^·− production and the effect of exogenously supplied ROS. A transient increase in extracellular O₂^·− production by the embryonic axes preceded significant desiccation-induced viability loss. Thereafter, progressively more oxidizing intracellular conditions, as indicated by a significant shift in glutathione half-cell reduction potential, accompanied cell and axis death, coinciding with the disruption of nuclear membranes. Most hydrogen peroxide (H₂O₂)-dependent O₂^·− production was found in a cell wall fraction that contained extracellular peroxidases (ECPOX) with molecular masses of ∼50 kDa. Cinnamic acid was identified as a potential reductant required for ECPOX-mediated O₂^·− production. H₂O₂, applied exogenously to mimic the transient ROS burst at the onset of desiccation, counteracted viability loss of sub-lethally desiccation-stressed seeds and of excised embryonic axes grown in tissue culture. Hence, extracellular ROS produced by embryonic axes appear to be important signalling components involved in wound response, regeneration and growth.