Physical mapping of human insulin-like growth factor-I using specific monoclonal antibodies.

The primary structure of recombinant human (h) insulin-like growth factor-I (IGF-I) epitopes recognized by a panel of 28 monoclonal antibodies (mAbs) is characterized. Pairwise mAb epitope mapping defines eight 'epitopic clusters' (I-VIII) which cover nearly the entire solvent-exposed IGF-I surface. Monoclonal antibody reactivity with 32 overlapping synthetic peptides and with IGF-I mutants is used to associate these epitopic clusters with the probable primary IGF-I sequences recognized. Epitopic cluster I involves residues in the C-domain and the first alpha-helix of the A-domain; clusters II, V and VII involve principally the B-domain; clusters III and IV map to amino acid sequences (55-70) and (1-13) respectively; cluster VI includes the A- and B-domains; and cluster VIII involves mainly the C-terminal part of the B-domain. Data indicate that this mAb panel defines 14 distinct IGF-I epitopes. The specific inhibition of HEL 92.1.7 IGF-I-promoted proliferation by these mAbs was explored. Direct correlation between mAb affinity and inhibitory activity was observed except in the case of clusters III- and VIII-specific mAbs. Finally, the combination of epitopic cluster I and II mAbs detect 0.5-10 ng/ml hIGF-I in a sandwich immunoassay, with no IGF-II crossreactivity. These anti-IGF-I mAbs are, therefore, useful for both the inhibition of IGF-I mitogenic activity and for the quantification of this growth factor. The potential use of this mAb panel in tumor cell growth control is discussed.

Biotransformation of terodiline. III. Opposed stereoselectivity in the benzylic and aromatic hydroxylations in rat liver microsomes.

1. Terodiline (N-tert-butyl-4,4-diphenyl-2-butylamine) is a racemic drug with anticholinergic and/or calcium antagonistic activity, which is subject to renewed interest as a potential remedy for urinary incontinence. As part of the current investigations on terodiline, the metabolism of its enantiomers is being investigated. 2. The metabolism of the enantiomers of terodiline in rat liver microsomes is slow, as for the racemate, though the S-enantiomer is metabolized more rapidly than its optical antipode. Phenobarbitone pretreatment of the rats enhances the metabolism with a marked increase in the conversion of the S-enantiomer. 3. While aromatic p-hydroxylation greatly exceeds benzylic oxidation in the metabolism of R-terodiline, this situation is reversed in the metabolism of S-terodiline. Moreover, the rate of aromatic p-hydroxylation of racemic terodiline follows that of R-terodiline, while the rate of benzylic hydroxylation of racemic terodiline follows that of S-terodiline. Phenobarbital pretreatment of the rats had little or no effect on aromatic p-hydroxylation but markedly increased benzylic oxidation. 4. Separation of the mixture of p-hydroxylated metabolites into diastereomeric pairs showed that their composition is highly dependent on which form of terodiline is used as substrate. 5. The results from the study are compatible with the participation of multiple forms of cytochrome P-450 enzymes.

A simplified radioimmunoassay for melatonin and its application to biological fluids. Preliminary observations on the half-life of plasma melatonin in man.

A simplified and rapid radioimmunoassay (RIA) for melatonin is presented. Melatonin is extracted from seru, plasma or urine and RIA is performed by using [3H]melatonin as the tracer. The standard curve covers the range 0.2--4.3 nmol/l. By increasing the sample volume the range can be extended to 0.06 nmol/l. The intra-assay variability is 7% (relative standard deviation = rsd) and the inter-assay variability is 10% (rsd). The recovery of melatonin added to calf serum is 96%. The long term variability of the assay (43 assays on aliquots of one serum sample during 6 months) is 13.5% (rsd). The serum levels in man after one oral dose of 430 mumol melatonin have been measured. The peak value, 620 nmol/l, was noted after 0.5 h and the melatonin concentration was still above the normal range at 24 h (2.1 nmol/l).

Melatonin in rat pineal gland and serum; rapid parallel decline after light exposure at night.

Melatonin content in the rat pineal decreases at night after exposure of animals to light, from 20 to 2 pmol/gland in 15 min. Melatonin concentration in serum fell precipitously in a manner almost identical to the drop in pineal melatonin only with a 5 min time lag. It is concluded that the rapid decrease in the pineal melatonin content is mainly due to an inhibited synthesis of melatonin.