Growth hormone in urine: development of an ultrasensitive assay applicable to plasma and urine

A radiometric assay for human growth hormone (HGH) was developed based on a polyclonal goat anti-HGH antiserum covalently coupled to nonsedimenting polyacrylamide particles. HGH can be specifically immunoextracted from sample volumes of up to 10 ml. Subsequently, bound HGH is identified and quantitatively measured by a 125I-labelled monoclonal anti-HGH antibody. The assay is insensitive to plasma proteins from 10 to greater than 90%, to changing NaCl and urea molarities and to pH ranges from 6 to 8. The sensitivity in the second incubation is 2 pg/tube, corresponding to a maximum sensitivity of 300 fg/ml of a sample volume of 10 ml (urine) or of 40 pg/ml, if a volume of 50 microliter (plasma) is assayed. In healthy children, a mean HGH excretion of 6.5 ng/24 h was found with a large interindividual range from undetectable to 37.4 ng. An important intraindividual night-to-night variation of HGH excretion was found in several subsequent first morning void samples in healthy children. The mean excretion in 13 HGH-deficient children was 0.9 ng/24 h off therapy and increased to a mean of 6.9 ng/24 h on therapy. In acromegalic patients, the excreted HGH amounted to 73-208 ng/24 h. Preliminary results suggest that the ultrasensitive assay applied to plasma and urine could be a considerable improvement of diagnosis and follow-up of disorders of HGH secretion.     

Regulation of DNA synthesis and capacity for initiation in DNA temperature mutants of Escherichia coli. III. Synthesis of the dnaA protein and of DNA-binding proteins

Summary The synthesis and action of the dnaA product with respect to DNA initiation and the synthesis of DNA-binding proteins in Escherichia coli was examined. Results indicate that when dnaA product is irreversibly denatured and must be synthesized before initiation can occur, its synthesis and action appear to be complete approximately 30 min before initiation takes place. However, in mutants whose dnaA product is temperature reversible the action of the dnaA product appears to occur near the time of initiation. Examination of the DNA-binding proteins from the mutants suggests that a 53 kd protein, possibly the dnaA product, may be synthesized at the time of initiation under normal conditions at permissive temperature. The presence of active dnaA product appears to trigger the synthesis of a 60–65 kd protein which may be responsible for preventing another immediate initiation event.