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.     

Na+/K+-ATPase Inhibition Partially Mimics the Ethanol-Induced Increase of the Golgi Cell-Dependent Component of the Tonic GABAergic Current in Rat Cerebellar Granule Cells

Cerebellar granule cells (CGNs) are one of many neurons that express phasic and tonic GABAergic conductances. Although it is well established that Golgi cells (GoCs) mediate phasic GABAergic currents in CGNs, their role in mediating tonic currents in CGNs (CGN-I_tonic) is controversial. Earlier studies suggested that GoCs mediate a component of CGN-I_tonic that is present only in preparations from immature rodents. However, more recent studies have detected a GoC-dependent component of CGN-I_tonic in preparations of mature rodents. In addition, acute exposure to ethanol was shown to potentiate the GoC component of CGN-I_tonic and to induce a parallel increase in spontaneous inhibitory postsynaptic current frequency at CGNs. Here, we tested the hypothesis that these effects of ethanol on GABAergic transmission in CGNs are mediated by inhibition of the Na⁺/K⁺-ATPase. We used whole-cell patch-clamp electrophysiology techniques in cerebellar slices of male rats (postnatal day 23–30). Under these conditions, we reliably detected a GoC-dependent component of CGN-I_tonic that could be blocked with tetrodotoxin. Further analysis revealed a positive correlation between basal sIPSC frequency and the magnitude of the GoC-dependent component of CGN-I_tonic. Inhibition of the Na⁺/K⁺-ATPase with a submaximal concentration of ouabain partially mimicked the ethanol-induced potentiation of both phasic and tonic GABAergic currents in CGNs. Modeling studies suggest that selective inhibition of the Na⁺/K⁺-ATPase in GoCs can, in part, explain these effects of ethanol. These findings establish a novel mechanism of action of ethanol on GABAergic transmission in the central nervous system.     

Chromosome transfer and R-prime formation by an RP4::mini-Mu derivative in Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, and Proteus mirabilis

We have introduced into the wide host range conjugative plasmid RP4, a mini-Mu derivative which was known to be able to transpose spontaneously in E. coli K-12, and to induce in such a host several kinds of chromosomal rearrangements including replicon fusions. Unlike RP4, RP4::mini-Mu can mediate the transfer of the host chromosome to a recipient bacterium and generate R primes at high frequencies (10^?4 for the transfer of a given marker, 10^?5 for the formation of R primes carrying a given marker). Two such RP4::mini-Mu plasmids were introduced into one Salmonella typhimurium strain, one Klebsiella pneumoniae strain, and one Proteus mirabilis strain. Each of these three strains were mated with an E. coli K-12 recipient and transconjugants carrying R primes were recovered in all three cases at frequencies ranging from 5 × 10^?6 to 10^?7.     

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.     

Hexapeptide analogue of somatostatin, Sandoz 201-456. Conformational study in dimethylsulfoxide by 1D and 2D n.m.r. methods

The conformational properties of the somatostatin analogue 201-456 (1) have been studied by high field n.m.r. in DMSO. This analogue is the base structure of nine derivates synthesized by Bauer et al. and shows a very low biological activity, although derived structures such as SMS 201-995 (2) are very potent. Our study has shown an important difference between the most stable conformation of the two compounds: although the beta turn type II' structure at the Phe3-Trp4-Lys5 level is present in both analogues, an important conformational change appears at the cystine bridge. In SMS 201-995 the beta turn/beta sheet conformation is stabilized by the additional amino-acids D-Phe1 and Thr8 (ol) through intramolecular H-bonds.     

Calcium-stimulated myofibrillar ATPase activity correlates with shortening velocity of muscle fibres in Xenopus laevis

Summary The iliofibularis muscle ofXenopus laevis is reported to contain five types of fibres which have different force—velocity relationships. Ten fibres of each type were selected on the basis of succinate dehydrogenase activity, cross-sectional area and location in the muscle, in order to assess the validity of the fibre type classification.Maximum calcium-stimulated myofibrillar ATPase activity (V _max) and apparent Michaelis constant (K _m) for ATP were determined for these 50 fibres from serial sections. The values obtained varied according to the type of fibre. Type 1 had the highest and type 5 the lowest values forK _m andV _max.In a separate experiment, single freeze-dried fibres were used to determine the relationship between their ATP content and apparentK _m for ATP. There was a tendency for high ATP concentrations in fibres with highK _m values.When myofibrillar ATPase activity was related to the maximum velocity of shortening of the five fibre types, a significant correlation was found. It is concluded that calcium-stimulated myofibrillar ATPase histochemistry allows an estimate of the maximum shortening velocity of muscle fibres fromXenopus laevis.