Clinical chronopharmacology of ACTH 1-17. I. Effects on plasma cortisol and urinary 17-hydroxycorticosteroids

The aim of the investigation was to study the effects of ACTH 1-17 on both plasma cortisol and urinary 17-OHCS in health adult young males with regard to the time (clock hours) at which this polypeptide was injected. Eight healthy adults (males from 18-30 years) volunteered for the study. They were synchronized with a diurnal activity from 0700 to 0000 and a nocturnal rest. Each week, during 6 consecutive weeks (January 19 to February 25, 1980), a 3-day test was performed on Saturday, Sunday and Monday. On Sundays 3 control-tests and 3 ACTH-tests were programmed during which either saline or 100 micrograms ACTH 1-17 were injected i.m. at respectively 0700, 1400 and 2100. During each 3 day-test (72 h) the urinary excretion of 17-OHCS was determined every 4 h at fixed clock hours. In addition, on Sundays, venous blood was sampled prior to control or ACTH injections at respectively 0700, 1400, and 2100 and 20, 40, 60, 90, 120, 150 and 180 min thereafter. Plasma cortisol (radioimmunoassay) was determined in samples thus collected. Both conventional and cosinor methods were used for statistical analyses. A strong and statistically significant rise of plasma cortisol was observed after all of the ACTH 1-17 injections. The obtained mean response curves were observed after all of the ACTH 1-17 injections. The obtained mean response curves were similar in form and parallel. The highest plasma cortisol curve corresponded to ACTH injected at 0700, the lowest to ACTH injected at 2100. The curve corresponding to ACTH injected at 1400 went in-between. The 24-h urinary excretion of 17-OHCS after ACTH 1-17 was approximately 4 times greater than the control value when injected at 0700, approximately 3 times greater than control when injected at 1400 and only twice greater than control when injected at 2100. In terms of changes in plasma cortisol and 17-OHCS the greatest best benefit of ACTH 1-17 is achieved when this polypeptide is injected at 0700, rather than at 1400 or 2100 in diurnally active subjects.     

Characterization of competent cells and early events of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

The insertion of foreign DNA in plants occurs through a complex interaction between Agrobacteria and host plant cells. The marker gene β-glucuronidase of Escherichia coli and cytological methods were used to characterize competent cells for Agrobacterium-mediated transformation, to study early cellular events of transformation, and to identify the potential host-cell barriers that limit transformation in Arabidopsis thaliana L. Heynh. In cotyledon and leaf explants, competent cells were mesophyll cells that were dedifferentiating, a process induced by wounding and-or phytohormones. The cells were located either at the cut surface or within the explant after phytohormone pretreatment. In root explants, competent cells were present in dedifferentiating pericycle, and were produced only after phytohormone pretreatment. Irrespective of their origin, the competent cells were small, isodiametric with thin primary cell walls, small and multiple vacuoles, prominent nuclei and dense cytoplasm. In both cotyledon and root explants, histological enumeration and β-glucuronidase assays showed that the number of putatively competent cells was increased by preculture treatment, indicating that cell activation and cell division following wounding were insufficient for transformation without phytohormone treatment. Exposure of explants for 48 h to A. tumefaciens produced no characteristic stress response nor any gradual loss of viability nor cell death. However, in the competent cell, association between the polysaccharide of the host cell wall and that of the bacterial filament was frequently observed, indicating that transformation required polysaccharide-to-polysaccharide contact. Flow cytofluorometry and histological analysis showed that abundant transformation required not only cell activation (an early state exhibiting an increase in nuclear protein) but also cell proliferation (which in cotyledon tissue occurred at many ploidy levels). Noncompetent cells could be made competent with the appropriate phytohormone treatments before bacterial infection: this should aid analysis of critical steps in transformation procedures and should facilitate developing new strategies to transform recalcitrant plants.