Determination of three-dimensional structures of proteins by simulated annealing with interproton distance restraints. Application to crambin, potato carboxypeptidase inhibitor and barley serine proteinase inhibitor 2

An automated method, based on the principle of simulated annealing, is presented for determining the three-dimensional structures of proteins on the basis of short (less than 5 A) interproton distance data derived from nuclear Overhauser enhancement (NOE) measurements. The method makes use of Newton's equations of motion to increase temporarily the temperature of the system in order to search for the global minimum region of a target function comprising purely geometric restraints. These consist of interproton distances supplemented by bond lengths, bond angles, planes and soft van der Waals repulsion terms. The latter replace the dihedral, van der Waals, electrostatic and hydrogen-bonding potentials of the empirical energy function used in molecular dynamics simulations. The method presented involves the implementation of a number of innovations over our previous restrained molecular dynamics approach [Clore, G.M., Brünger, A.T., Karplus, M. and Gronenborn, A.M. (1986) J. Mol. Biol., 191, 523-551]. These include the development of a new effective potential for the interproton distance restraints whose functional form is dependent on the magnitude of the difference between calculated and target values, and the design and implementation of robust and fully automatic protocol. The method is tested on three systems: the model system crambin (46 residues) using X-ray structure derived interproton distance restraints, and potato carboxypeptidase inhibitor (CPI; 39 residues) and barley serine proteinase inhibitor 2 (BSPI-2; 64 residues) using experimentally derived interproton distance restraints. Calculations were carried out starting from the extended strands which had atomic r.m.s. differences of 57, 38 and 33 A with respect to the crystal structures of BSPI-2, crambin and CPI respectively. Unbiased sampling of the conformational space consistent with the restraints was achieved by varying the random number seed used to assign the initial velocities. This ensures that the different trajectories diverge during the early stages of the simulations and only converge later as more and more interproton distance restraints are satisfied. The average backbone atomic r.m.s. difference between the converged structures is 2.2 +/- 0.3 A for crambin (nine structures), 2.4 +/- 0.3 A for CPI (eight structures) and 2.5 +/- 0.2 A for BSPI-2 (five structures). The backbone atomic r.m.s. difference between the mean structures derived by averaging the coordinates of the converged structures and the corresponding X-ray structures is 1.2 A for crambin, 1.6 A for CPI and 1.7 A for BSPI-2.     

Influence of hirudin on the consumption of antithrombin III in experimental DIC

The effect of hirudin and heparin on thrombin-induced consumption of antithrombin III, fibrinogen and platelets was studied in a rat model. Antithrombin III is consumed by tolerated thrombin doses by about 20 per cent. Hirudin and heparin ameliorate the consumption of fibrinogen and platelets at the low thrombin dose used. At high thrombin doses, tolerated only during simultaneous administration of exogenous inhibitors, heparin leads to markedly increased consumption of anti-thrombin III, whereas hirudin does not. With either kind of treatment, the thrombin effect on fibrinogen and platelets is inhibited, however, hirudin acts independently of a cofactor in contrast to heparin.     

Serum immunoassay of a small cell lung cancer associated ganglioside: development of a sensitive scintillation proximity assay

We here report an enzyme linked immunosorbent assay (ELISA) and a scintillation proximity assay (SPA) for detection of the ganglioside FucGM₁ in sera from small cell lung cancer (SCLC) patients. The SPA was more sensitive and reproducible than the ELISA. In this assay, monoclonal antibodies specific for FucGM₁ were bound to SPA particles and incubated with labelled FucGM₁ and 100 µl test-serum overnight, and counted in a -counter. The sensitivity was 0.2 ng. Seven out of twenty sera from SCLC patients were positive, whereas none of twenty sera from healthy individuals were positive for FucGM₁. The SPA was more sensitive than the previously reported HPTLC as well as a direct ELISA.Abbreviations MAb monoclonal antibody - SPA scintillation proximity assay - HPTLC high performance thin layer chromatography - SCLC small cell lung cancer - FucGM₁ Fuc1-2Gal1-3GalNAc1-4(NeuAc2-3)-Gal1-4Glc1-1Cer - ELISA enzyme linked immunosorbent assay - FCS foetal calf serum - PBS phosphate buffered saline     

Sequence of the 5-aminolevulinic acid dehydratase-encoding gene from the hyperthermophilic methanogen, Methanothermus sociabilis.

We report on the sequence of the Methanothermus sociabilis aladh gene, which encodes the 5'-aminolevulinic acid dehydratase. The identity of the enzyme was determined by sequence comparison and by expression in Escherichia coli.     

Expression, isolation and purification of antibody fragments fused to maltose-binding protein in Escherichia coli]

We have fused the variable domains of a mouse antibody to the C-terminal end of the maltose-binding protein (malE), at the genetic level. The hybrid proteins were expressed in E. coli under control of the malEp promoter, and exported to the periplasm, at low temperature. They were purified by affinity chromatography on cross-linked amylose. When the two variable domains were fused together through a peptide link, the hybrid displayed similar affinity and specificity to the antigen as the native antibody.     

Interactions between freshwater snails and tadpoles: competition and facilitation

Summary Freshwater snails and anuran tadpoles have been suggested to have their highest population densities in ponds of intermediate size where abiotic disturbance (e.g. desiccation) is low and large predators absent. Both snails and tadpoles feed on periphytic algae and, thus, there should be a large potential for competitive interactions to occur between these two distantly related taxa. In a field experiment we examined the relative strength of competition between two closely related snail species, Lymnaea stagnalis and L. peregra, and between L. stagnalis and tadpoles of the common frog, Rana temporaria. Snail growth and egg production and tadpole size at and time to metamorphosis were determined. Effects on the common food source, periphyton, were monitored with the aid of artificial substrates. Periphyton dry weight was dramatically reduced in the presence of snails and/or tadpoles. There were no competitive effects on growth or egg production of the two snail species when they were coexisting. Mortality of L. peregra was high (95%) after reproduction, but independent of treatment. Growth of L. stagnalis was reduced only at the highest tadpole densities, whereas egg production was reduced both by intraspecific competition and by competition with tadpoles. Differences in egg production were retained after tadpole metamorphosis. Tadpole larval period increased, weight of metamorphosing frogs decreased and growth rate was reduced as a function of increasing tadpole density. However, contrary to expectation, snails had a positive effect on tadpole larval period, weight and growth rate. Further, in experimental containers without snails there was a dense growth of the filamentous green alga Cladophora sp. We suggest that the facilitative effects of snails on tadpoles are due to an indirect mutualistic mechanism, involving competition between food sources of different quality (microalgae and Cladophora sp.) and tadpoles being competitively dominant over snails for the preferred food source (microalgae). In the presence of tadpoles snails will be forced to feed on low-quality Cladophora, increasing nutrient turnover rates, which results in enhanced productivity of microalgae, increasing tadpole food resources. Thus, tadpoles have a negative effect on snails through resource depression, while snails facilitate tadpole growth through an indirect enhancement of food availability.     

The effect of a single colony of the red wood ant,Formica polyctena,on the spider fauna (Araneae) of a beech forest floor

Summary The predation on spiders in a forest ecosystem by a colony of red wood ants, Formica polyctena, was estimated using a barrier to isolate the colony. Of the ants' total prey, 4.6% were spiders. In order to estimate the effect of F. polyctena within their hunting area on the spider population, the spiders' population density was studied inside and outside the hunting area. Samples of the forest floor were taken, spider webs were counted and pitfall traps were used. No significant difference was found in density or composition of the spider fauna inside and outside the hunting area.     

Inhibition of the transthylakoid gradient of electrochemical proton potential by the local anesthetic dibucaine

The effects of the local anesthetic dibucaine on coupling between electron transport and ATP synthesis-hydrolysis by the coupling-factor complex (CF₀CF₁ ATPase) were investigated in thylakoid membranes from Spinacia oleracea L. cv. Monatol. Evidence is presented that inhibition of ATP synthesis was produced by a specific uncoupling mechanism which was based on dibucaine-membrane surface interactions rather than on the interaction of dibucaine with the ATPase complex. Dibucaine reduced the osmotic space of thylakoid vesicles. At low pH of the medium it stimulated ATP hydrolysis beyond the rates obtained with optimum concentrations of ‘classical’ uncouplers. After addition of dibucaine, there was displacement of membrane-bound Mg²⁺ and strong thylakoid stacking in the presence of only low Mg²⁺ concentrations. Inhibition of ATP synthesis and transmembrane pH gradient increased with medium pH. Hydrolysis of ATP by isolated CF₁ and the CF₀CF₁ complex was only slightly affected by dibucaine. The data are discussed assuming the involvement of localized proton channels on the membrane surface in protonic coupling of electron transport and ATP synthesis. A hypothesis for the mechanisms of action of local anesthetics at the thylakoid membrane is presented.     

An integrated approach to oilseed rape cultivar selection using phenotypic stability

Summary Various methods of evaluating phenotypic stability have been proposed; however, no single method can adequately describe cultivar performance. The objectives of this study were to integrate a number of methods of evaluating stability and to use this approach for cultivar selection. These objectives were considered in the context of the broad-based oilseed rape cultivar (Brassica napus spp. oleifera) evaluation system currently used in western Canada. Regression analysis was used to assess cultivar response to environments. Cluster analysis was used to assemble cultivars into groups with similar regression coefficients (b _i ) and mean yield. Three parametric stability parameters, years within locations mean square (MS; Y/L), Shukla's stability variance ( _i ² ), and Francis and Kannenberg's coefficient of variability (CV _i ), were compared to determine which method would be most suitable for selection of oilseed rape cultivars from within clustered groups. Yield data from three cultivars and six breeding lines that had been tested for 2 years at 26 locations in the Western Canola Cooperative Test A were used for all calculations. The cluster analysis was successful in identifying commercially acceptable breeding lines. The parameter MS _i Y/L was considered to be more appropriate than either CV _i or _i ² , because it measured only the unpredictable portion of the genotype x environment interaction and was independent of the other cultivars in the test. The use of cluster analysis to group entries with similar b _i values and mean yields, followed by selection for stability within groups, was advocated.Contribution No. 846 of the Plant Science Department, University of Manitoba