An H-bond between two residues from different loops of the acetylcholine binding site contributes to the activation mechanism of nicotinic receptors

The molecular mechanisms of nicotinic receptor activation are still largely unknown. The crystallographic structure of the acetylcholine binding protein (AChBP) reveals a single H-bond between two different acetylcholine binding loops. Within these homologous loops we systematically introduced alpha4 residues into the alpha7/5HT(3) chimeric receptor and found that the single point mutations G152K (loop B) and P193I (loop C) displayed a non-additive increase of equilibrium binding affinity for several agonists compared with the double mutant G152K/P193I. In whole-cell patch-clamp recordings, G152K, P193I and G152K/P193I mutants displayed an increase up to 5-fold in acetylcholine potency with a large decrease of the apparent Hill coefficients (significantly smaller than one). Concomitantly, the G152K/P193I mutant showed a dramatic loss of high-affinity alpha-bungarotoxin binding (100-fold decrease), thus pinpointing a new contact area for the toxin. Fitting the data with an allosteric-kinetic model, together with molecular dynamic simulations, suggests that the presence of the inter-backbone H-bond between positions 152 and 193, revealed in alpha4 and in alpha7 double mutant but not in alpha7, coincides with a large stabilization of both open and desensitized states of nicotinic receptors.     

EPR studies of in vivo radical production by lipopolysaccharide: potential role of iron mobilized from iron-nitrosyl complexes

Although oxidative stress has been implicated in the pathogenesis of sepsis, there is little evidence for the formation of radicals other than nitric oxide in its experimental models. Here we used low temperature EPR and EPR spin trapping to monitor nitric oxide and secondary radical formation in blood, liver, and bile samples from rats treated with a low lipopolysaccharide (LPS) dose (0.25 mg) and with dimethyl sulfoxide (DMSO) and the spin trap alpha-(4-pyridyl 1-oxide)- N-t-butylnitrone (POBN). The results showed that production of secondary radicals triggered by LPS is delayed in regard to maximum nitric oxide synthesis and is iron-dependent. One of the secondary produced radicals was identified as the hydroxyl radical. Its formation is proposed to occur because of the mobilization of redox-active iron required to repair the nitrosyl complexes produced by LPS. The results suggest that iron chelation may be a useful adjuvant therapy for treating sepsis.     

Mollusc larval shell formation: amorphous calcium carbonate is a precursor phase for aragonite

The larval shells of the marine bivalves Mercenaria mercenaria and Crassostrea gigas are investigated by polarized light microscopy, infrared spectroscopy, Raman imaging spectroscopy, and scanning electron microscopy. Both species contain similar shell ultrastructures. We show that larval shells contain amorphous calcium carbonate (ACC), in addition to aragonite. The aragonite is much less crystalline than non-biogenic aragonite. We further show that the initially deposited mineral phase is predominantly ACC that subsequently partially transforms into aragonite. The postset juvenile shell, as well as the adult shell of Mercenaria also contains aragonite that is less crystalline than non-biogenic aragonite. We conclude that ACC fulfills an important function in mollusc larval shell formation. It is conceivable that ACC may also be involved in adult shell formation.     

Egg distributions and the information a solitary parasitoid has and uses for its oviposition decisions

Approximately three decades ago the question was first answered whether parasitoids are able to assess the number or origin of eggs in a host for a solitary parasitoid, Leptopilina heterotoma, by fitting theoretically derived distributions to empirical ones. We extend the set of different theoretically postulated distributions of eggs among hosts by combining searching modes and abilities in assessing host quality. In the models, parasitoids search either randomly (Poisson) (1) or by vibrotaxis (Negative Binomial) (2). Parasitoids are: (a) assumed to treat all hosts equally, (b) able to distinguish them in unparasitised and parasitised hosts only, (c) able to distinguish them by the number of eggs they contained, or (d) able to recognise their own eggs. Mathematically tractable combinations of searching mode (1 and 2) and abilities (a,b,c,d) result in seven different models (M1a, M1b, M1c, M1d, M2a, M2b and M2c). These models have been simulated for a varying number of searching parasitoids and various mean numbers of eggs per host. Each resulting distribution is fitted to all theoretical models. The model with the minimum Akaike's information criterion (AIC) is chosen as the best fitting for each simulated distribution. We thus investigate the power of the AIC and for each distribution with a specified mean number of eggs per host we derive a frequency distribution for classification.Firstly, we discuss the simulations of models including random search (M1a, M1b, M1c and M1d). For M1a, M1c and M1d the simulated distributions are correctly classified in at least 70% of all cases. However, in a few cases model M1b is only properly classified for intermediate mean values of eggs per host. The models including vibrotaxis as searching behaviour (M2a, M2b and M2c) cannot be distinguished from those with random search if the mean number of eggs per host is low. Among the models incorporating vibrotaxis the three abilities are detected analogously as in models with random search.Experiments with two species of solitary parasitoids (L. heterotoma and Asobara tabida) are conducted. All theoretically postulated distributions are separately fitted to the resulting experimental egg distributions. The AIC criterion is used to choose the best fitting theoretical distribution. For both parasitoid species the frequency distribution of best fitting models for experimental data is compared to the classification of distributions generated by simulations. This leads to the conclusion that both L. heterotoma and A. tabida are able to distinguish between parasitised and unparasitised hosts. For L. heterotoma the results point to an ability to assess the number of eggs in a host, whereas A. tabida does not seem to have this ability. This difference suggests that an egg is more valuable for L. heterotoma than for A. tabida.     

Molecular phylogeny of Larrea and its allies (Zygophyllaceae): reticulate evolution and the probable time of creosote bush arrival to North America.

Nucleotide sequences of Rubisco Large Subunit (rbcL) and the internal transcribed spacers (ITS) of nrDNA were obtained for the five species of Larrea and one species each of Bulnesia (ITS only) and Plectrocarpa (rbcL only). Parsimony analyses were conducted, including sequences from seven genera of Zygophyllaceae reported by other authors-Kallstroemia, Zygophyllum, Augea, Fagonia, Pintoa, Guaiacum, and Porlieria. The main conclusions of the present study are (1) the Argentine endemic Plectrocarpa tetracantha belongs to the subfamily Larreoideae (New World Clade); (2) all three phylogenies obtained from rbcL, ITS, and combined data sets show a close relationship between the tetraploid L. cuneifolia (sect. Bifolium) and the diploid multifoliolate pair L. nitida-L. ameghinoi (sect. Larrea), which could result from a possible intersectional hybrid origin of the tetraploid; (3) L. divaricata (sect. Bifolium) and L. tridentata (sect. Bifolium) form a highly supported monophyletic group, which agrees with previous cytogenetic and molecular evidence; and (4) the rate of nucleotide substitution of rbcL was estimated based on geological and fossil records. Under the molecular clock hypothesis, nucleotide sequence divergence between L. divaricata and L. tridentata suggests a Late Neogene (8.4 to 4.2 mybp) time of arrival of the diploid ancestors of L. tridentata to North American deserts.     

Characterization of bimodal chronotype and its association with sleep: A population-based study

The circadian system coordinates internal events in a daily schedule to make sure that the body systems are synchronized to environmental time and internal cues. One important behavioral aspect of the circadian system is the chronotype. It is usually assessed through subjective questionnaires, being the Horne-Ostberg Morningness–Eveningness Questionnaire (MEQ) one of the most used. It classifies individuals into three major categories: morning, evening, and intermediate types. Recently, it has been hypothesized the existence of a fourth chronotype, the bimodal type, through an algorithm derived from the MEQ responses. Bimodals answer as morning-types in some questions, and as evening-types in others, resulting in an intermediate total score. To better characterize this phenotype, the present study aimed to detect and characterize the frequency of the bimodal chronotype in the EPISONO, a large population-based cohort, as well as to verify the association between bimodality and sleep parameters and genetic variation in the PER3 gene. Of the 1,042 individuals who participated of the EPISONO, 857 had MEQ filled correctly. We found that 16% of our sample were bimodal types. We observed that bimodal individuals were significantly younger and had lower body mass index. The association between PER3 VNTR genotype and gender with bimodal chronotype was not significant. However, we found an association between bimodality and Epworth Sleepiness Scale (EES) and apnea-hypopnea index (AHI). We did not find a statistically significant difference between bimodals and intermediate non-bimodals for the studied variables. Lastly, it was observed that the most significant predictors for bimodal chronotype were female gender, AHI, and EES. In conclusion, the present work provides more evidence that the bimodal type might have to be considered when classifying chronotype and its association with young age and sleepiness may be due to the influence of social and environmental factors.