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.     

Interferon inhibits DNA Synthesis induced in Mouse Lymphocyte Suspensions by Phytohaemagglutinin or by Allogeneic Cells

IN addition to its well known antiviral activity, interferon has recently been shown to inhibit the multiplication of tumour and mammalian cells in cell culture^1–6. We report here the inhibition by interferon of DNA synthesis induced in mouse spleen lymphocytes by the non-viral stimuli phytohaemagglutinin (PHA) and allogeneic lymphocytes. These findings are in accord with our contention that interferon affects cell function and, furthermore, they suggest that by acting on lymphocytes, interferon plays a role in the immunological response of the host.     

Penicillin-binding proteins 4 and 5 of Haemophilus influenzae are involved in cell wall incorporation

Abstract Permeabilized cells of Haemophilus influenzae incorporate wall precursors into murein material in an ampicillin-sensitive reaction. In resistant transformants that contain the low antibiotic affinity penicillin-binding proteins (PBPs) 4 and 5, the sensitivity of this incorporation reaction to ampicillin is proportionally lower, suggesting a catalytic role for these proteins in wall synthesis. We conclude that, analogous to the reaction in Escherichia coli , PBPs 4 and 5 of H. influenzae have transpeptidase activity.     

Improved micro-method for the high-performance liquid chromatographic determination of caffeine and paraxanthine in biological fluids

A high-performance liquid chromatographic procedure is reported for reproducibly and sensitively quantitating caffeine and its N-demethylated metabolite paraxanthine in micro-samples. A 5-μm reversed-phase radial compression column and 214-nm fixed wavelength ultraviolet detector were used to attain a sensitivity sufficient to quantitate these compounds at concentratios as low as 80 ng/ml using only 25 μl of sample. The assay is applicable to microliter samples of whole blood, serum, plasma, saliva, amniotic, cerebro-spinal and gastric fluids such as might be obtained in studies involving small animals or neonates. The utility of the assay is illustrated with caffeine and paraxanthine levels measured in several maternal and fetal fluids following constant-rate intravenous infusion of caffeine into a rabbit throughout pregnancy.     

Compartmentation of high-energy phosphates in resting and beating heart cells

The subcellular distribution of ATP, ADP, creatine phosphate and creatine has been analyzed by fast detergent fractionation of isolated frog heart cells. Digitonin fractionation (0.5 mg/ml, 10 s at 2 degrees C in 20 mM 4-morpholinepropanesulfonic acid/3 mM EDTA/230 mM mannitol medium) was used to separate mitochondria and myofilaments from cytosol. To separate myofilaments from the other cellular compartments. Triton X-100 was used (2%, 15 s in the same medium as digitonin). For either resting or beating cells the total cellular contents of ATP, ADP, creatine phosphate and creatine was similar, nevertheless the O2 consumption was 6-times higher. The compartmentation of these metabolites was also identical. Myofilaments contain 1.1 nmol ADP per mg total cellular proteins. In the cytosolic compartment the metabolite concentrations, all measured in nmol per mg total cellular proteins, were: ATP, 13; ADP, 0.25-0.05; creatine phosphate, 18.5 and creatine, 14. This indicated that the reaction catalyzed by creatine kinase was in a state of (or near) equilibrium.     

Effects of antimicrotubular agents in cAMP production and in steroidogenic response of isolated rat Leydig cells

In dispersed rat Leydig cells, colchicine was found to stimulate basal cAMP production and testosterone secretion in a dose and time-dependent manner, but to a lesser extent than LH. However, these drugs are unable to stimulate adenylate cyclase activity in plasma membranes isolated from these cells. The amount of testosterone secreted at 150 min under the influence of colchicine and LH added simultaneously was not different from the amount produced during stimulation by LH alone. It is only after exposure of the cells for 1 hr to colchicine that the accumulation of cAMP in response to LH was inhibited; furthermore, both intracellular and medium testosterone accumulation in response to the hormone were reduced. Similar effects were observed with two other alkaloids, vinblastine and podophyllotoxin. The three drugs also inhibited the stimulation of testosterone secretion by 8-Br-cAMP or choleratoxin. These studies suggest that the state of microtubule polymerization and/or tubulin can influence the process of steroidogenesis in rat Leydig cells.     

Decomposition of three halophytes in different habitats of an eastern scheldt salt marsh

Summary A 20.5-month study was undertaken to determine detrital processing of the halophytesSpartina anglica, Elytrigia pungens, andHalimione portulacoides in three different habitats of an estuarine salt marsh in the South-West Netherlands. Decomposition was measured using litter-bags of three different mesh sizes to partition the effects of different faunal groups on decomposition. From April 1980 through October 1981 litter-bags were sampled regulary from a creek, the upper marsh, and from a plant-debris belt on the higher marsh. Dry weights and nutritive values were measured and animals were counted. Mainly rates of loss are reported here. Zonal differences were significant. At first, decomposition in the creek was most rapid. After two months the processes in the creek slowed down because of the trapping of silt by the bags, which probably simulated the natural course of the decomposition process in the water. Decomposition on the marsh followed the most regular pattern, while in the plant-debris belt the pattern was very irregular. Population dynamics of microfaunal organisms supported these findings. In the plant-debris belts loss rates seem to be higher than on the marsh, because of the influence of detritivorous macrofaunal organisms. The loss rates of the three plant species differed significantly.Halimione decomposed fastest, especially in the beginning, and in the plant-debris habitat. On the upper marsh and in the plant-debris belt the loss rates ofSpartina seem to be a little higher than those ofElytrigia.Communication No. 233, Delta Institute for Hydrobiological Research, Yerseke, The Netherlands.