The use of existing environmental networks for the post-market monitoring of GM crop cultivation in the EU.

The European Union (EU) Directive 2001/18/EC on the deliberate release of genetically modified organisms (GMOs) into the environment requires that both Case-Specific Monitoring (CSM) and General Surveillance (GS) are considered as post-market implementing measures. Whereas CSM is directed to monitor potential adverse effects of GMOs or their use identified in the environmental risk assessment, GS aims to detect un-intended adverse effects of GMOs or their use on human and animal health or the environment. Guidance documents on the monitoring of genetically modified (GM) plants from the Commission and EFSA clarify that, as appropriate, GS can make use of established routine surveillance practices. Networks involved in routine surveillance offer recognised expertise in a particular domain and are designed to collect information on important environmental aspects over a large geographical area. However, as the suitability of existing monitoring networks to provide relevant data for monitoring impacts of GMOs is not known, plant biotechnology companies developed an approach to describe the processes and criteria that will be used for selecting and evaluating existing monitoring systems. In this paper, the availability of existing monitoring networks for this purpose is evaluated. By cataloguing the existing environmental monitoring networks in the EU, it can be concluded that they can only be used, in the context of GMO cultivation monitoring, as secondary tools to collect baseline information.

Reef-coral proteins as visual, non-destructive reporters for plant transformation.

Recently, five novel fluorescent proteins have been isolated from non-bioluminescent species of reef-coral organisms and have been made available through ClonTech. They are AmCyan, AsRed, DsRed, ZsGreen and ZsYellow. These proteins are valuable as reporters for transformation because they do not require a substrate or external co-factor to emit fluorescence and can be tested in vivo without destruction of the tissue under study. We have evaluated them in a large range of plants, both monocots and dicots, and our results indicate that they are valuable reporting tools for transformation in a wide variety of crops. We report here their successful expression in wheat, maize, barley, rice, banana, onion, soybean, cotton, tobacco, potato and tomato. Transient expression could be observed as early as 24 h after DNA delivery in some cases, allowing for very clear visualization of individually transformed cells. Stable transgenic events were generated, using mannose, kanamycin or hygromycin selection. Transgenic plants were phenotypically normal, showing a wide range of fluorescence levels, and were fertile. Expression of AmCyan, ZsGreen and AsRed was visible in maize T1 seeds, allowing visual segregation to more than 99% accuracy. The excitation and emission wavelengths of some of these proteins are significantly different; the difference is enough for the simultaneous visualization of cells transformed with more than one of the fluorescent proteins. These proteins will become useful tools for transformation optimization and other studies. The wide variety of plants successfully tested demonstrates that these proteins will potentially find broad use in plant biology.

Production of stably transformed cassava plants via particle bombardment.

A novel protocol, based on biolistics and regeneration via organogenesis, was developed for genetic transformation of cassava (Manihot esculenta Crantz). The in vitro performance of cassava cultivars CMC40, MPer183 and MCol22 was evaluated, and the regeneration protocol was modified to improve shoot production from explants for transformation experiments. Somatic cotyledons were used as a target tissue in the transformation experiments using the Particle Inflow Gun and a plasmid containing the uidA gene in transient assays. The effect of different parameters for particle bombardment efficiency, including the amount of DNA used, the flying distance of the projectiles and the pre- and post-plasmolysis time of the target tissue, was evaluated and the conditions were partially optimised. Stably transformed cassava plants of cvs. MCol22 and TMS60444 were produced using the partially optimised conditions and two different vector constructs carrying the hpt gene as the selectable marker. The selection protocol was optimised further, and a rooting test was developed for screening the regenerants for antibiotic resistance to reduce the number of escapes obtained after primary selection. The production of stably transformed cassava lines and the expression of the transgenes was verified by Southern blot analysis and RT-PCR.

Sodium space and intravascular volume: dietary sodium effects in cystic fibrosis and healthy adolescent subjects.

OBJECTIVE: To assess the physiologic response to salt depletion in subjects with cystic fibrosis (CF) and control male adolescents for sodium balance, sodium space, and stimulation of the renin-angiotensin-aldosterone axis. DESIGN: Seven subjects with CF and six controls received a salt-replete (150 or 290 mmol NaCl per day) diet and then a salt-deplete (10 mmol NaCl per day) diet while in a clinical research center. RESULTS: Space maintenance: CF subjects responded to salt depletion with a greater weight loss than did controls (1.9 vs 0.8 kg) and a decrease in 24Na+ space, whereas controls maintained 24Na+ space. Paired (Na-deplete/Na-replete) blood volumes decreased in subjects with CF, but not in controls. Renin-angiotensin-aldosterone axis stimulation: During salt repletion, subjects with CF had significantly higher aldosterone values than did controls in the afternoon, but not at 7:00 AM. During salt depletion, plasma renin activity and aldosterone increased significantly more in subjects with CF than in controls (renin, 35 vs 13 ng/mL/hour [9.7 vs 3.6 ng.L-1 s-1]; aldosterone: 181 vs 101 ng/dL [5021 vs 2802 pmol/L]). Furthermore, the angiotensin antagonist saralasin increased renin much more in subjects with CF (154 vs 36 ng/mL per hour [43 vs 10 ng.L-1 s-1]). Vasomotor functions: Mean arterial pressure was decreased in subjects with CF on both diets and decreased significantly more with low salt only in subjects with CF. During salt depletion, subjects with CF showed enhanced orthostatic tolerance (less heart rate increase with standing) compared with controls, thus obscuring their volume loss. The blood pressure response to an acute infusion of saralasin suggested that in salt-replete subjects with CF, but not in controls, angiotensin receptors were functional in maintaining vascular tone. During salt depletion, angiotensin was more important for maintenance of blood pressure in subjects with CF than in controls, because the saralasin-induced drop in blood pressure was 20%, ie, close to shock levels, in subjects with CF, and only 6% in controls. CONCLUSION: The data suggest that patients with CF are so successful in compensating for volume depletion by vigorous activation of the renin-angiotensin system that salt depletion/dehydration cannot be recognized easily by routine clinical measurements, eg, capillary refill, serum sodium levels, or tachycardia.

Developmental sequelae of long-term infant tracheostomy.

The records of 130 children who had had a tracheostomy for more than one month between 1972 and 1982 were reviewed for birth, medical and demographic characteristics, and 32 who were not severely physically or mentally handicapped were followed up at a mean age of 5.5 years. Rates of mortality and morbidity were high, and half of the survivors had moderate to severe intellectual and physical impairments. Even those with the most optimal outcome had significant developmental problems, including slower growth-rate and higher than normal incidences of behavior problems and speech difficulties. Most survivors needed special educational intervention or rehabilitative therapies.

Inhibition of amiloride-sensitive sodium conductance by indoleamines.

To examine a possible role of indoleamines in the regulation of epithelial sodium absorption, the effect of serotonin (5-hydroxytryptamine) and several derivatives on electrolyte transport was measured in vitro in the baboon bronchus and in the trachea and colon of sodium-deficient rats. Serotonin, melatonin (N-acetyl-5-hydroxytryptamine), and harmaline (1-methyl-7-methoxy-3,4-dihydro-beta-carboline) inhibited sodium transport in all three preparations in a similar manner to the natriuretic agent amiloride. In all three epithelia, sodium absorption via the amiloride-sensitive pathway constitutes a substantial portion of total electrolyte transport, measured as the amiloride-sensitive short-circuit current. Thus 25 microM amiloride inhibited the short-circuit current 21% in the rat trachea, 63% in the baboon bronchus, and 90% in the rat colon. Serotonin, melatonin, and harmaline inhibited the amiloride-sensitive portion of the short-circuit current from the luminal side of the epithelium. The inhibition was rapid, requiring only seconds, and maximal inhibition by serotonin was identical to that by amiloride. When sodium was omitted from the luminal solution, the short-circuit current was reduced a similar amount, suggesting that sodium absorption was being inhibited by both amiloride and the indoles. The IC50 value for amiloride was 50 nM in the baboon bronchus and 500 nM in the rat colon. In contrast, the IC50 value for serotonin was 0.4 mM in the baboon bronchus and 8 mM in the rat colon. These results, together with the wide distribution of amine-precursor-uptake-and-decarboxylation (APUD) cells in the respiratory and intestinal tract, suggest that certain indoleamines could play a role as local regulators of fluid and electrolyte transport. For example, in the airways, indoleamines may be one of the factors involved in regulation of the depth of the periciliary fluid layer.