Expression of two heterologous promoters, Agrobacterium rhizogenes rolC and cauliflower mosaic virus 35S, in the stem of transgenic hybrid aspen plants during the annual cycle of growth and dormancy

We monitored, for the first time, the activity of two model heterologous promoters, the Agrobacterium rhizogenes rolC and the cauliflower mosaic virus (CaMV) 35S, throughout the annual cycle of growth and dormancy in a perennial species, hybrid aspen. Each promoter was fused to the uidA -glucuronidase (GUS) reporter gene and the constructs were introduced into the hybrid aspen genome by Agrobacterium-mediated transformation. Both wildtype and transgenic plants were cultivated under different regimes of photoperiod and temperature to induce passage through one growth-dormancy-reactivation cycle, and at intervals GUS staining was assessed in stem sections. In rolC::uidA transformants, GUS activity in rapidly growing current-year shoots was not only tissue-specific, being localized to the phloem, but also cell-specific at the shoot base, where it was present only in the companion cells. However, during the onset of dormancy induced by short photoperiod, GUS activity shifted laterally from the phloem to include the cortex and pith. After subsequent exposure to chilling temperatures to induce the transition between the dormancy stages of rest and quiescence, GUS activity almost disappeared from all stem tissues, but regained its original phloem specificity and intensity after the shoots were reactivated by exposing them to long photoperiod and high temperatures. In contrast, GUS activity in the stem of 35S::uidA transformants was strong in all tissues except for the vascular cambium and xylem, and did not vary in intensity during the growth-dormancy-reactivation cycle. The lateral shift and increased intensity of GUS activity in the stem of rolC::uidA transformants during dormancy induction was shown to be associated with the accumulation of starch, and to be mimicked by incubating stem sections in sucrose, as well as glucose and fructose, but not sorbitol, prior to the GUS assay. Our results demonstrate that the activities of the rolC and 35S promoters varied in very different, unpredictable ways during the annual cycle of growth and dormancy in a perennial species, and indicate that the spatial and temporal variation in rolC promoter activity that we observed in the stem of transgenic hybrid aspen plants is attributable to cellular and seasonal changes in sucrose content.     

The Robustness of Recombination Frequency Estimates in Intercrosses with Dominant Markers

The robustness of the maximum likelihood estimates of recombination frequencies has been investigated in double intercrosses with complete dominance at both loci. The robustness was investigated with respect to bias in the recombination frequency estimates due to: (1) limited sample sizes, (2) heterogeneity in recombination frequencies between sexes or among meioses and (3) factors that distort the segregation-misclassification or differential viability. In the coupling phase, the recombination frequency estimates are quite robust with respect to most of the investigated factors. Potentially, the most serious cause of a bias is misclassifications, which tend to increase the recombination frequency estimates. In the repulsion phase, misclassifications are particularly serious, leading to extreme discrepancies between true and observed values. In addition, limited sample size and sex differences in recombination can also bias recombination frequency estimates in repulsion. These effects may pose serious problem in genetic mapping with random amplified polymorphic DNA (RAPD) markers.     

Synthesis of α-mannosylated ergot alkaloids employing α-mannosidase

The ergot alkaloids elymoclavine, ergometrine and chanoclavine were α-mannosylated with α-mannosidase as catalyst. The kinetic reaction with p-nitrophenyl α-mannoside as glycosyl donor gave ca 28 % yield of chanoclavine α-mannoside, whereas the equilibrium reaction with mannose as the glycosyl donor gave ca 11 % yield. However, in the case of elymoclavine and ergometrine, higher yields of α-mannosides were obtained with the equilibrium approach (18 and 13 %).     

Effects of energy deprivation on the fly pupil mechanism: evidence for a rigor state

The energy dependence of the pupil pigment-migrations in the fly Musca domestica was studied in live animals, using optical techniques and nitrogen-gas induced anoxia. The results obtained can be summarized in 3 points:

1. Energy deficiency can make the pupil mechanism stop in any state, extreme or intermediate.
2. Anoxia induced during intermittent stimulation makes the pupil stop in the closed state (aggregated pigment granules).
3. During long-term anoxia the pupil very slowly opens (dispersal of pigment granules), irrespective of ambient intensity.

The slow anoxic opening (point 3) is more than 1000 times slower than that predicted for free diffusion of pigment granules in water. Assuming realistic values of cytoplasm viscosity, this implies that anoxia causes the pigment granules to attach to rigid structures in the cells, in analogy with the rigor state in anoxic muscles. The rigor phenomenon in the pupil mechanism prevents experimental discrimination between active and passive processes of pigment migration. Normal pupil opening has a time course which agrees reasonably with a passive diffusion process, but it is argued that an active transportation of granules away from the rhabdom is more likely in the dark adapted eye.     

Hormonal Characterization of Transgenic Tobacco Plants Expressing the rolC Gene of Agrobacterium rhizogenes TL-DNA

Transgenic tobacco (Nicotiana tabacum L. cv Wisconsin 38) plants expressing the Agrobacterium rhizogenes rolC gene under the control of the cauliflower mosaic virus 35S RNA promoter were constructed. These plants displayed several morphological alterations reminiscent of changes in indole-3-acetic acid (IAA), cytokinin, and gibberellin (GA) content. However, investigations showed that neither the IAA pool size nor its rate of turnover were altered significantly in the rolC plants. The biggest difference between rolC and wild-type plants was in the concentrations of the cytokinin, isopentenyladenosine (iPA) and the gibberellin GA19. Radio-immunoassay and liquid chromatography-mass spectrometry measurements revealed a drastic reduction in rolC plants of iPA as well as in several other cytokinins tested, suggesting a possible reduction in the synthesis rate of cytokinins. Furthermore, gas chromatography-mass spectrometry quantifications of GA19 showed a 5- to 6-fold increase in rolC plants compared with wild-type plants, indicating a reduced activity of the GA19 oxidase, a proposed regulatory step in the gibberellin biosynthesis. Thus, we conclude that RolC activity in transgenic plants leads to major alterations in the metabolism of cytokinins and gibberellins.     

Whole inactivated SIV vaccine grown on human cells fails to protect against homologous SIV grown on simian cells

Several groups have reported protection against experimental SIV infection in macaques immunized with a whole inactivated virus vaccine. The aim of the current study was to investigate whether five macaques vaccinated with whole inactivated SIV and previously shown to be protected against challenge with two divergent strains of SIV grown on human cells could resist challenge with a subsequent homologous SIV grown on macaque cells. We show here that this same vaccine did not protect when the challenge virus was grown on primary cells of monkey origin.     

Sediment toxicity in the Kattegat and Skagerrak

Sediments were sampled from 62 sites in the Kattegat and Skagerrak, which are located between the Baltic and the North Sea in the Western Atlantic, during autumn 1989 and spring 1990. From each site 5 to 6 samples were taken wit ha box-corer. After mixing to composite samples on board, transport and storage (at 4 °C for 2 to 4 weeks), the samples were tested for toxicity to Daphnia magna and Nitocra spinipes. Immobility in Daphnia after exposure to 16 percent sediment (wet wt) in reconstituted standardized water (ISO, 1982) ranged from 0 to 88 percent after 24 h and from 3 to 95 percent after 48 h. For Nitocra the toxicity, determined as the 96-h LC50 (% wet wt) at 7 salinity, ranged from > > 32 percent (nontoxic) to 1.8 percent (most toxic). All exposures were made in duplicates and the effects obtained in the duplicates with the same sediment were correlated to each other. However, sediment toxicity to Daphnia and Nitocra was not. The test with Nitocra, which was made at several concentrations of sediment, was considered to give the most reliable picture of sediment toxicity in the Kattegat and Skagerrak. This ambient toxicity assessment identified three areas with toxic sediment, (1) the Göta älv estuary (outside the city of Göteborg) and its surroundings, (2) the Bay of Laholm in southern Kattegat, which is an area with periodic oxygen depletion and where repeated mussel kills have occurred during the last decade, and (3) an area in the open Skagerrak northwest of Skagen (the tip of the Jutland peninsula). Sediments, which had been stored at 4 °C, were tested again after 6 to 13 mos with the Nitocra test. Stored sediment toxicity was poorly correlated with fresh sediment toxicity. The average detoxification during storage was 5 times, but the range was 3 orders of magnitude, from 17 times more toxic to 73 times less toxic. The reasons for the observed areal and storage differences in sediment toxicity are so far not understood.     

Interactive effects of rising temperatures and urbanisation on birds across different climate zones: A mechanistic perspective

Climate change and urbanisation are among the most pervasive and rapidly growing threats to biodiversity worldwide. However, their impacts are usually considered in isolation, and interactions are rarely examined. Predicting species' responses to the combined effects of climate change and urbanisation, therefore, represents a pressing challenge in global change biology. Birds are important model taxa for exploring the impacts of both climate change and urbanisation, and their behaviour and physiology have been well studied in urban and non-urban systems. This understanding should allow interactive effects of rising temperatures and urbanisation to be inferred, yet considerations of these interactions are almost entirely lacking from empirical research. Here, we synthesise our current understanding of the potential mechanisms that could affect how species respond to the combined effects of rising temperatures and urbanisation, with a focus on avian taxa. We discuss potential interactive effects to motivate future in-depth research on this critically important, yet overlooked, aspect of global change biology. Increased temperatures are a pronounced consequence of both urbanisation (through the urban heat island effect) and climate change. The biological impact of this warming in urban and non-urban systems will likely differ in magnitude and direction when interacting with other factors that typically vary between these habitats, such as resource availability (e.g. water, food and microsites) and pollution levels. Furthermore, the nature of such interactions may differ for cities situated in different climate types, for example, tropical, arid, temperate, continental and polar. Within this article, we highlight the potential for interactive effects of climate and urban drivers on the mechanistic responses of birds, identify knowledge gaps and propose promising future research avenues. A deeper understanding of the behavioural and physiological mechanisms mediating species' responses to urbanisation and rising temperatures will provide novel insights into ecology and evolution under global change and may help better predict future population responses.     

Clinical pharmacology of the new COMT inhibitor CGP 28 014

CGP 28 014 is a specific inhibitor of catechol-O-methyltransferase (COMT) in vivo. In humans, the inhibition was assessed by measuring urinary excretion of isoquinolines and with the levodopa test. Following administration of CGP 28 014, urinary excretion of isoquinolines was significantly increased. In rats, CGP 28 014 reduced plasma and striatal concentrations of 3-O-methyldopa (30MD) in a dose-dependent manner. Acute and subchronic administration of CGP 28 014 alone or in combination with the peripherally acting decarboxylase inhibitor benserazide decreased plasma 30MD as an index of COMT inhibition by about 50%. There seems to be a close relationship between the time-course of plasma concentrations of CGP 28 014 and the extent of COMT inhibition assessed by the 30MD/DOPA ratio in plasma.