Modelling of the disulphide-swapped isomer of human insulin-like growth factor-1: implications for receptor binding.

Insulin-like growth factor-1 (IGF-1) is a serum protein which unexpectedly folds to yield two stable tertiary structures with different disulphide connectivities; native IGF-1 [18-61,6-48,47-52] and IGF-1 swap [18-61,6-47, 48-52]. Here we demonstrate in detail the biological properties of recombinant human native IGF-1 and IGF-1 swap secreted from Saccharomyces cerevisiae. IGF-1 swap had a approximately 30 fold loss in affinity for the IGF-1 receptor overexpressed on BHK cells compared with native IGF-1.The parallel increase in dose required to induce negative cooperativity together with the parallel loss in mitogenicity in NIH 3T3 cells implies that disruption of the IGF-1 receptor binding interaction rather than restriction of a post-binding conformational change is responsible for the reduction in biological activity of IGF-1 swap. Interestingly, the affinity of IGF-1 swap for the insulin receptor was approximately 200 fold lower than that of native IGF-1 indicating that the binding surface complementary to the insulin receptor (or the ability to attain it) is disturbed to a greater extent than that to the IGF-1 receptor. A 1.0 ns high-temperature molecular dynamics study of the local energy landscape of IGF-1 swap resulted in uncoiling of the first A-region alpha-helix and a rearrangement in the relative orientation of the A- and B-regions. The model of IGF-1 swap is structurally homologous to the NMR structure of insulin swap and CD spectra consistent with the model are presented. However, in the model of IGF-1 swap the C-region has filled the space where the first A-region alpha-helix has uncoiled and this may be hindering interaction of Val44 with the second insulin receptor binding pocket.     

Determination of duodenogastric reflux by quantification of bile acids in the gastric juice

The existence of duodeno-gastric reflux was evaluated in a group of 15 healthy subjects, in fasting and for 24 hours. The assessment of duodeno-gastric reflux was made by quantitation of the bile acids (BA) present in the gastric juice. The individual free and conjugated BA were separated and quantified by means of thin-layer chromatography and in situ spectrofluorometry. In 7 of the subjects studied no BA were detected, and in the other 8 subjects the BA levels were below 6 mumol reflux/hour. There were no free BA detected in any of the subjects. The levels of BA in gastric juice increased progressively with age, but there were no differences between sex. The chromatographic technique used is highly sensitive for the analysis of BA in biological samples. The study of BA in the gastric juice provides a quantitative and reliable assessment of the degree of duodeno-gastric reflux.     

Cytogenetic analysis of mouse metaphase II oocytes following exposure to tritiated water

Female mice were given different dosages (0, 3.0, 7.5, 15.0, or 30 muCi/ml) of tritium in their drinking water continuously from 3 to 7 weeks of age to assess the effects on germ cell chromosomes. At 8-9 weeks of age, mice were superovulated and metaphase II oocytes were processed and C-banded for cytogenetic analyses. Chromatid acentric fragments were the only type of structural aberration detected, and their incidence was higher in controls than in any of the tritiated water (HTO) groups. Analysis of numerical chromosomal aberrations revealed that the incidence of hypoploid (N = 19) oocytes was higher in oocytes from mice who drank HTO as compared with controls. However, the effects of HTO upon aneuploidy induction was not definitive due to the increase the incidence of aberrations in mouse oocytes can be related to the low dose rate resulting from chronic HTO exposure and possibly death of tritium-damaged cells.     

The effect of storage on the frequency of dominant lethals in Drosophila melanogaster

Summary Ethylenimine, ethyl methanesulfonate and formaldehyde have been shown to produce a storage effect in dominant lethality in Drosophila melanogaster.