Practical applications of time-averaged restrained molecular dynamics to ligand-receptor systems: FK506 bound to the Q50R,A95H,K98I triple mutant of FKBP-13

Summary The ability of time-averaged restrained molecular dynamics (TARMD) to escape local low-energy conformations and explore conformational space is compared with conventional simulated-annealing methods. Practical suggestions are offered for performing TARMD calculations with ligand-receptor systems, and are illustrated for the complex of the immunosuppressant FK506 bound to Q50R,A95H,K98I triple mutant FKBP-13. The structure of ¹³C-labeled FK506 bound to triple-mutant FKBP-13 was determined using a set of 87 NOE distance restraints derived from HSQC-NOESY experiments. TARMD was found to be superior to conventional simulated-annealing methods, and produced structures that were conformationally similar to FK506 bound to wild-type FKBP-12. The individual and combined effects of varying the NOE restraint force constant, using an explicit model for the protein binding pocket, and starting the calculations from different ligand conformations were explored in detail.Abbreviations DG distance geometry - dmFKBP-12 double-mutant (R42K,H87V) FKBP-12 - FKBP-12 FK506-binding protein (12 kDa) - FKBP-13 FK506-binding protein (13 kDa) - HSQC heteronuclear single-quantum coherence - K_NOE force constant (penalty) for NOE-derived distance restraints - MD molecular dynamics - NOE nuclear Overhauser effect - SA simulated annealing - TARMD molecular dynamics with time-averaged restraints - tmFKBP-13 triple-mutant (Q50R,A95H,K98I) FKBP-13 - wtFKBP-12 wild-type FKBP-12     

The elongation of amylose and amylopectin chains in isolated starch granules

The aim of this work was to investigate the conditions required for amylose synthesis in starch granules. Although the major granule-bound isoform of starch synthase - GBSSI - catalyses the synthesis of amylose in vivo, ¹⁴C from ADP[¹⁴C]glucose was incorporated primarily into a specific subset of amylopectin chains when supplied to starch granules isolated from pea (Pisum sativum L.) embryos and potato (Solanum tuberosum L.) tubers. Incubation of granules with soluble extracts of these organs revealed that the extracts contained compounds that increased the incorporation of ¹⁴C into amylose. These compounds were rendered inactive by treatment of the extracts with α-glucosidase, suggesting that they were malto-oligosaccharides. Consistent with this idea, provision of pure malto-oligosaccharides to isolated granules resulted in a dramatic shift in the pattern of incorporation of ¹⁴C, from amylopectin chains to amylose molecules. Comparison of the pattern of incorporation in granules from wild-type peas and lam mutant peas which lack GBSSI showed that this effect of malto-oligosaccharides was specifically on GBSSI. The significance of these results for understanding of the synthesis of amylose and amylopectin in storage organs is discussed.     

Effects of Interactions BetweenPasteurella haemolyticaandBordetella parapertussisonin vitroPhagocytosis by Lung Macrophages

The aim of the work was to determine the effect of exposing ovine bronchoalveolar macrophages (BAM)in vivotoPasteurella haemolyticaand/orBordetella parapertussison the subsequent uptake and killing ofP. haemolyticaby these cellsin vitro. Exposurein vivotoP. haemolyticadid not affect the uptake ofP. haemolyticaby BAMin vitrobut reduced (P< 0·05) the intracellular killing of bacteria. Exposurein vivotoB. parapertussishad no significant effect on either the uptake of killing ofP. haemolytica in vitro. However, sequential exposurein vivotoB. parapertussisandP. haemolyticareduced both the ingestion (P< 0·05) and killing (P< 0·001) ofP. haemolytica in vitro. These results indicate that exposure toP. haemolyticacompromised the bacterial killing mechanisms of BAM and that synergy betweenB. parapertussisandP. haemolyticareduced the ability of BAM to ingest bacteria.     

Production, Purification, and Characterization of Recombinant Human Hemoglobin Rainier Expressed inSaccharomyces cerevisiae

Hemoglobin Rainier is a naturally occurring hemoglobin variant in which the β145 tyrosine is substituted with cysteine. The α and β^Rainierglobin cDNAs were cloned in a high copy number vector and expressed inSaccharomyces cerevisiaeunder the control of galactose-regulated hybrid promoters. Using this system, we have expressed individual α and β^Rainierglobin chains. Coexpression of both α and β^RainiercDNAs resulted in the production of a functional hemoglobin molecule. Purification of the recombinant protein was accomplished by ion exchange chromatography. The N-termini of the α and β chains were correctly processed, and the molecular mass, as determined by mass spectrometry, indicated amino acid composition identical to that of natural hemoglobin Rainier. The chromatographic properties of the recombinant hemoglobin Rainier were similar to human-derived hemoglobin A₀. The purified recombinant hemoglobin molecule was shown to have an elevated oxygen affinity and a reduced cooperativity as previously reported for natural hemoglobin Rainier. Production of recombinant hemoglobin and especially hemoglobin variants like hemoglobin Rainier has the potential to facilitate use of hemoglobin as a blood substitute as well as in specific applications, such as for use as a therapeutic agent in the treatment of hypotension associated with septic shock.     

The mechanism of resistance to paraquat is strongly temperature dependent in resistant Hordeum leporinum Link and H. glaucum Steud.

Paraquat-resistant biotypes of the closely-related weed species Hordeum leporinum Link and H. glaucum Steud. are highly resistant to paraquat when grown during the normal winter growing season. However, when grown and treated with paraquat in summer, these biotypes are markedly less resistant to paraquat. This reduced resistance to paraquat in summer is primarily a result of increased temperature following herbicide treatment. The mechanism governing this decrease in resistance at high temperature was examined in H. leporinum. No differences were observed between susceptible and resistant biotypes in the interaction of paraquat with isolated thylakoids when assayed at 15, 25, or 35 °C. About 98 and 65% of applied paraquat was absorbed through the leaf cuticle of both biotypes at 15 and 30 °C, respectively. Following application to leaves, more herbicide was translocated in a basipetal direction in the susceptible biotype compared to the resistant biotype at 15 °C. However, at 30 °C more paraquat was translocated in a basipetal direction in the resistant biotype. Photosynthetic activity of young leaf tissue from within the leaf sheath which had not been directly exposed to paraquat was measured 24 h after treatment of plants with para. quat. This activity was inhibited in the susceptible biotype when plants were maintained at either 15 °C or 30 °C after treatment. In contrast, photosynthetic activity of such tissue of the resistant biotype was not inhibited when plants were maintained at 15 °C after treatment, but was inhibited at 30 °C. The mechanism of resistance in this biotype of H. leporinum correlates with decreased translocation of paraquat and decreased penetration to the active site. This mechanism is temperature sensitive and breaks down at higher temperatures.We are grateful to Zeneca Agrochemicals, Jealotts Hill, Berkshire, UK who provided [¹⁴C]paraquat. E.P. was supported through a Ph.D. scholarship from the Australian International Development Assistance Bureau and C.P. was the recipient of an Australian Research Council Postdoctoral Fellowship.     

Contrasting leaf and ‘ecosystem’ CO2 and H2O exchange in Avena fatua monoculture: Growth at ambient and elevated CO2

Elevated CO₂ (ambient + 35 Pa) increased shoot dry mass production in Avena fatua by 68% at maturity. This increase in shoot biomass was paralleled by an 81% increase in average net CO₂ uptake (A) per unit of leaf area and a 65% increase in average A at the ecosystem level per unit of ground area. Elevated CO₂ also increased ecosystem A per unit of biomass. However, the products of total leaf area and light-saturated leaf A divided by the ground surface area over time appeared to lie on a single response curve for both CO₂ treatments. The approximate slope of the response suggests that the integrated light saturated capacity for leaf photosynthesis is 10-fold greater than the ecosystem rate. Ecosystem respiration (night) per unit of ground area, which includes soil and plant respiration, ranged from-20 (at day 19) to-18 (at day 40) mol m^-2 s^-1 for both elevated and ambient CO₂ Avena. Ecosystem below-ground respiration at the time of seedling emergence was -10 mol m^-2 s^-1, while that occuring after shoot removal at the termination of the experiment ranged from -5 to-6 mol m^-2 s^-1. Hence, no significant differences between elevated and ambient CO₂ treatments were found in any respiration measure on a ground area basis, though ecosystem respiration on a shoot biomass basis was clearly reduced by elevated CO₂. Significant differences existed between leaf and ecosystem water flux. In general, leaf transpiration (E) decreased over the course of the experiment, possibly in response to leaf aging, while ecosystem rates of evapotranspiration (ET) remained constant, probably because falling leaf rates were offset by an increasing total leaf biomass. Transpiration was lower in plants grown at elevated CO₂, though variation was high because of variability in leaf age and ambient light conditions and differences were not significant. In contrast, ecosystem evapotranspiration (ET) was significantly decreased by elevated CO₂ on 5 out of 8 measurement dates. Photosynthetic water use efficiencies (A/E at the leaf level, A/ET at the ecosystem level) were increased by elevated CO₂. Increases were due to both increased A at leaf and ecosystem level and decreased leaf E and ecosystem ET.     

Seasonal flooding,nitrogen mineralization and nitrogen utilization in a prairie marsh

Flooding can be an important control of nitrogen (N) biogeochemistry in wetland ecosystems. In North American prairie marshes, spring flooding is a dominant feature of the physical environment that increases emergent plant production and could influence N cycling. I investigated how spring flooding affects N availability and plant N utilization in whitetop (Scolochloa festucacea) marshes in Manitoba, Canada by comparing experimentally spring-flooded marsh inside an impoundment with adjacent nonflooded marsh. The spring-flooded marsh had net N mineralization rates up to 4 times greater than nonflooded marsh. Total growing season net N mineralization was 124 kg N ha^–1 in the spring-flooded marsh compared with 62 kg N ha^–1 in the nonflooded marsh. Summer water level drawdown in the spring-flooded marsh decreased net N mineralization rates. Net nitrification rates increased in the nonflooded marsh following a lowering of the water table during mid summer. Growing season net nitrification was 33 kg N ha^–1 in the nonflooded marsh but < 1 kg N ha^–1 in the spring-flooded marsh. Added NO₃ ^–1 induced nitrate reductase (NRA) activity in whitetop grown in pot culture. Field-collected plants showed higher NRA in the nonflooded marsh. Nitrate comprised 40% of total plant N uptake in the nonflooded marsh but <1% of total N uptake in the spring-flooded marsh. Higher plant N demand caused by higher whitetop production in the spring-flooded marsh approximately balanced greater net N mineralization. A close association between the presence of spring flooding and net N mineralization and net nitrification rates indicated that modifications to prairie marshes that change the pattern of spring inundation will lead to rapid and significant changes in marsh N cycling patterns.     

Synthesis and biological evaluation of novel NK-1 tachykinin receptor antagonists: The use of cycloalkyl amino acids as a template

In the course of a program aimed at synthesizing novel, potent NK-1 tachykinin receptor antagonists, we developed upon a bioactive model by comparing the low energy structures of a series of peptide and nonpeptide Substance P antagonists. The comparison was based on the super imposition of the aromatic rings, assuming that the rest of the molecule behaves predominantly as a template to arrange the key aromatic groups in the right spatial position. A series of 2-aminocyclohexane carboxylic acid analogues were then selected as the best templates for reproducing the postulated bioactive structure, leading to several pseudo-peptides with interesting biological activity. According to the molecular modeling, these compounds exhibit a neat parallel facing of the indolyl and naphthyl groups at about 3 Å distance. Ultraviolet absorption and steady state fluorescence measurements support this conclusion, showing a linear correlation between the spectral properties and the binding affinity of these analogues. Stacking of the indole ring with naphthalene gives rise to a complex characterized by a well-defined molar extinction coefficient. Consistently, steady state and lifetime fluorescence measurements suggest that the quenching process is ascribable to ground-state interactions between the chromophores. Implications of the π stacking propensity of aromatic groups in the biological activity of the compounds examined are briefly discussed. © 1995 John Wiley & Sons, Inc.