Probing the Flexibility of Tropomyosin and Its Binding to Filamentous Actin Using Molecular Dynamics Simulations

Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments.     

Stress responses and metal tolerance of <Emphasis Type="Italic">Chlamydomonas acidophila</Emphasis> in metal-enriched lake water and artificial medium

Chlamydomonas acidophila faces high heavy-metal concentrations in acidic mining lakes, where it is a dominant phytoplankton species. To investigate the importance of metals to C. acidophila in these lakes, we examined the response of growth, photosynthesis, cell structure, heat-shock protein (Hsp) accumulation, and metal adsorption after incubation in metal-rich lake water and artificial growth medium enriched with metals (Fe, Zn). Incubation in both metal-rich lake water and medium caused large decreases in photosystem II function (though no differences among lakes), but no decrease in growth rate (except for medium + Fe). Concentrations of small Hsps were higher in algae incubated in metal-rich lake-water than in metal-enriched medium, whereas Hsp60 and Hsp70A were either less or equally expressed. Cellular Zn and Fe contents were lower, and metals adsorbed to the cell surface were higher, in lake-water-incubated algae than in medium-grown cells. The results indicate that high Zn or Fe levels are likely not the main or only contributor to the low primary production in mining lakes, and multiple adaptations of C. acidophila (e.g., high Hsp levels, decreased metal accumulation) increase its tolerance to metals and permit survival under such adverse environmental conditions. Supposedly, the main stress factor present in the lake water is an interaction between low P and high Fe concentrations.     

Seed after-ripening and dormancy determine adult life history independently of germination timing

? Seed dormancy can affect life history through its effects on germination time. Here, we investigate its influence on life history beyond the timing of germination. ? We used the response of Arabidopsis thaliana to chilling at the germination and flowering stages to test the following: how seed dormancy affects germination responses to the environment; whether variation in dormancy affects adult phenology independently of germination time; and whether environmental cues experienced by dormant seeds have an effect on adult life history. ? Dormancy conditioned the germination response to low temperatures, such that prolonged periods of chilling induced dormancy in nondormant seeds, but stimulated germination in dormant seeds. The alleviation of dormancy through after-ripening was associated with earlier flowering, independent of germination date. Experimental dormancy manipulations showed that prolonged chilling at the seed stage always induced earlier flowering, regardless of seed dormancy. Surprisingly, this effect of seed chilling on flowering time was observed even when low temperatures did not induce germination. ? In summary, seed dormancy influences flowering time and hence life history independent of its effects on germination timing. We conclude that the seed stage has a pronounced effect on life history, the influence of which goes well beyond the timing of germination.     

Dual role of nNOS in ischemic injury and preconditioning

Background  

Nitric oxide (NO) is cardioprotective and a mediator of ischemic preconditioning (IP). Endothelial nitric oxide synthase (eNOS) is protective against myocardial ischemic injury and a component of IP but the role and location of neuronal nitric oxide synthase (nNOS) remains unclear. Therefore, the aims of these studies were to: (i) investigate the role of nNOS in ischemia/reoxygenation-induced injury and IP, (ii) determine whether its effect is species-dependent, and (iii) elucidate the relationship of nNOS with mitoK_ATP channels and p38MAPK, two key components of IP transduction pathway.     

Agonist stimulation of the serotonin1A receptor causes suppression of anoxia-induced apoptosis via mitogen-activated protein kinase in neuronal HN2-5 cells

Previous studies have indicated that stimulation of neuronal inhibitory receptors, such as the serotonin1A receptor (5-HT1A-R), could cause attenuation of the activity of both N-type Ca2+ channels and N-methyl-D-aspartic acid receptors, thus resulting in protection of neurons against excitotoxicity. The purpose of this study was to investigate if the 5-HT1A-R is also coupled to an alternative pathway that culminates in suppression of apoptosis even in cells that are deficient in Ca2+ channels. Using a hippocampal neuron-derived cell line (HN2-5) that is Ca2+ channel-deficient, we demonstrate here that an alternative pathway is responsible for 5-HT1A-R-mediated protection of these cells from anoxia-triggered apoptosis, assessed by deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL). The 5-HT1A-R agonist-evoked protection was eliminated in the presence of pertussis toxin and also required phosphorylation-mediated activation of mitogen-activated protein kinase (MAPK), as evidenced by the elimination of the agonist-elicited rescue of neuronal cells by the MAPK kinase inhibitor PD98059 but not by the phosphatidylinositol 3-kinase (PI-3K) inhibitor wortmannin. Furthermore, agonist stimulation of the 5-HT1A-R caused a 60% inhibition of anoxia-stimulated caspase 3-like activity in the HN2-5 cells, and this inhibition was abrogated by PD98059 but not by wortmannin. Although agonist stimulation of the 5-HT1A-R caused an activation of PI-3Kgamma in HN2-5 cells, our results showed that this PI-3Kgamma activity was not linked to the 5-HT1A-R-promoted regulation of caspase activity and suppression of apoptosis. Thus, in the neuronal HN2-5 cells, agonist binding to the 5-HT1A-R results in MAPK-mediated inhibition of a caspase 3-like enzyme and a 60-70% suppression of anoxia-induced apoptosis through a Ca2+ channel-independent pathway.     

Solvent extraction selection in the determination of isoflavones in soy foods

Acetonitrile is superior to acetone, ethanol and methanol in extracting the 12 phytoestrogenic soy isoflavone forms found in foods. At 53% organic solvent in water, raw soy flour, tofu, tempeh, textured vegetable protein and soy germ were evaluated for isoflavone extraction efficiency. The efficiency of acetonitrile extraction was demonstrated in mass balance evaluations of toasting of soy flour and soymilk heating.     

Stereochemistry of strictic acid and related furano-diterpenes from conyza japonica and grangea maderaspatana

Extraction of Conyza japonica gave strictic acid, ent-2β-hydroxy-15,16-epoxy-3,13(16),14-clerodatrien-18-oic acid and 5,7-dihydroxy-3,8,4′-trimethoxyflavone. Extraction of Grangea maderaspatana gave (-)-hardwickiic acid, ent-15,16-epoxy-1,3,13(16),14-clerodatetraen-18-oic acid and 3-hydroxy-8-acetoxypentadeca-1,9,14-trien-4,6-diyne. The structure of ent-2β-hydroxy-15,16-epoxy-3,13(16),14-cleroclatrien-18-oic acid was deduced by spectroscopic methods and by partial synthesis from (-)-hardwickiic acid and the stereochemistries of strictic acid and (ent-15,16-epoxy-1,3,13(16),14-clerodatraen-18-oic acid were established by correlation with ent-2β-hydroxy-15,16-epoxy-3,13(16),14-clerodatrien-18-oic acid.     

Molecular mapping of genes determining height, time to heading, and growth habit in barley (Hordeum vulgare).

A combination of random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers has been used to locate genes controlling important developmental characters in barley. The denso dwarfing gene has been mapped to the long arm of chromosome 3H. Stepwise multiple regression was also used to identify another region of the barley genome (on chromosome 7H), which contributed to variation in height. The denso locus was shown to be associated with delaying time to heading. A protein (WSP2) and an RAPD marker on barley chromosomes 5H and 6H, respectively, were also associated with time to heading. These results are discussed in relation to the genetic analysis of developmentally important traits and the development of dwarfing genes in barley breeding programs.     

Contraceptive failure: levels, trends and determinants in Matlab, Bangladesh

This study investigated the levels, trends and determinants of contraceptive use-failure in Matlab, Bangladesh, using a set of prospective data on 25,960 women of reproductive age. The data were extracted from the Record Keeping System (RKS) of Matlab for the period 1978-94. If there was any live birth during the use or within 7 months after the discontinuation of use, it was considered as a failure. The life table technique and hazard model were used as analytical tools. The results suggest that use-failure for pills, IUDs (TCu 200) and injectables and other temporary methods increased from 1978 to 1988, but began to decline after 1988. The cumulative probability of first-method failure within 1 year of method acceptance of the cohort of 1990-94 acceptors was 12.9% for pills, 2.0% for IUDs, 0.5% for injectables, 22.0% for condoms and 13.4% for 'other' methods (sampoon, foam, jelly and traditional methods). For pills, condoms and 'other' methods, the likelihood of failure declined with the duration of use; by contrast, the probability of an IUD failure increased over time, peaking at 3 years of use. The injectables maintained a low likelihood of failure regardless of the duration of use. The quality of Community Health Workers' (CHWs) performance was associated with the risk of failure of all temporary methods except condoms; women's background characteristics associated with failure varied by method. The effect of the quality of the CHWs' performance and the background variables on failure did not change much over time. It is felt that contraceptive failure deserves the serious attention of programme managers and policy makers to make the Bangladesh national family planning programme more successful.