Sensitive high-performance liquid chromatographic assay for pilocarpine in biological fluids using fluorescence derivatisation

A sensitive assay for pilocarpine in biological fluids has been developed involving HPLC of a fluorescent derivative of 4-bromomethyl-7-methoxycoumarin. Pilosine as internal standard was added before the derivatisation step. The fluorescent derivatives were well resolved and separated from excess reagent and endogeneous compounds on a cyanopropyl silica column. The detection limit of pilocarpine in biological fluids was 1.0 ng/ml and the assay was linear up to a concentration of 150 ng/ml. The assay was applied to a preliminary study of pilocarpine disposition in man after a single oral dose. This is the first report of pilocarpine excretion into saliva.     

A functional assessment of the response of grassland vegetation to reduced grazing and abandonment

Question: Predicting the impact of land‐use change on vegetation is vital to understanding how biodiversity and ecosystem function may respond. Is it correct to assume that abandonment is an extreme form of grazing reduction? Location: Borders and central Scotland. Methods: The analysis used data sets from two identical experiments where the impacts of two unfertilized, extensively grazed treatments and one unfertilized abandoned treatment were compared against the species dynamics of a pasture subject to normal, productive grazing management over a 16‐year period. Initial multivariate analysis using Principal Response Curves was used to assess if particular traits were associated with either extensive or abandoned treatments, and was checked using univariate tests of individual traits. RLQ analysis followed by clustering into response groups was used to assess if species behaved in a similar manner between sites. Results: For many traits/attributes the shift in value or proportion was approximately linear across the extensification treatments as grazing was removed. However, certain traits showed step changes and quadratic responses. Leaf dry matter content, an important effect trait, was in the latter group. Most traits/attributes and species behaved similarly at the two sites. However, traits such as regenerative strategy, seed length, longevity and mass and seed bank type behaved differently, indicating that they are not predictable response traits. Conclusion: The results indicate that responses to grazing removal during extensification are largely straightforward and largely independent of species pool. However, there are discrepancies that suggest that simple analyses of the impacts of land‐use changes such as grazing reduction may hide more complex responses.     

Neonatal MeCP2 is important for the organization of sex differences in vasopressin expression

Several neurodevelopmental disorders are marked by atypical Methyl-CpG-binding protein 2 (MeCP2) expression or function; however, the role of MeCP2 is complex and not entirely clear. Interestingly, there are sex differences in some of these disorders, and it appears that MeCP2 has sex-specific roles during development. Specifically, recent data indicate that a transient reduction in MeCP2 within developing amygdala reduces juvenile social play behavior in males to female-typical levels. These data suggest that MeCP2 within the amygdala is involved in programming lasting sex differences in social behavior. In the present study, we infused MeCP2 or control siRNA into the amygdala of male and female rats during the first three days of postnatal life in order to assess the impact of a transient reduction in MeCP2 on arginine vasopressin (AVP), a neural marker that is expressed differentially between males and females and is linked to a number of social behaviors. The expression of AVP, as well as several other genes, was measured in two-week old and adult animals. Two-week old males expressed more AVP and galanin mRNA in the amygdala than females, and a transient reduction in MeCP2 eliminated this sex difference by reducing the expression of both gene products in males. A transient reduction in MeCP2 also decreased androgen receptor (AR) mRNA in two-week old males. In adulthood, control males had more AVP-immunoreactive (AVP-ir) cells than females in the centromedial amygdala (CMA), bed nucleus of the striaterminalis (BST) and in the fibers that project from these cells to the lateral septum (LS). A transient reduction in MeCP2 eliminated this sex difference. Interestingly, there were no lasting differences in galanin or AR levels in adulthood. Reducing MeCP2 levels during development did not alter estrogen receptorα, neurofilament or Foxg1. We conclude that a transient reduction in MeCP2 expression in the developing male amygdala has a transient impact on galanin and AR expression but a lasting impact on AVP expression, highlighting the importance of MeCP2 in organizing sex differences in the amygdala.Key words: epigenetics, MeCP2, amygdala, sexual differentiation, development, arginine vasopressin, galanin     

Phylogeny of snakes (Serpentes): Combining morphological and molecular data in likelihood,Bayesian and parsimony analyses

Abstract

The phylogeny of living and fossil snakes is assessed using likelihood and parsimony approaches and a dataset combining 263 morphological characters with mitochondrial (2693 bp) and nuclear (1092 bp) gene sequences. The ‘no common mechanism’ (NCMr) and ‘Markovian’ (Mkv) models were employed for the morphological partition in likelihood analyses; likelihood scores in the NCMr model were more closely correlated with parsimony tree lengths. Both models accorded relatively less weight to the molecular data than did parsimony, with the effect being milder in the NCMr model. Partitioned branch and likelihood support values indicate that the mtDNA and nuclear gene partitions agree more closely with each other than with morphology. Despite differences between data partitions in phylogenetic signal, analytic models, and relative weighting, the parsimony and likelihood analyses all retrieved the following widely accepted groups: scolecophidians, alethinophidians, cylindrophiines, macrostomatans (sensu lato) and caenophidians. Anilius alone emerged as the most basal alethinophidian; the combined analyses resulted in a novel and stable position of uropeltines and cylindrophiines as the second‐most basal clade of alethinophidians. The limbed marine pachyophiids, along with Dinilysia and Wonambi, were always basal to all living snakes. Other results stable in all combined analyses include: Xenopeltis and Loxocemus were sister taxa (fide morphology) but clustered with pythonines (fide molecules), and Ungaliophis clustered with a boine‐erycine clade (fide molecules). Tropidophis remains enigmatic; it emerges as a basal alethinophidian in the parsimony analyses (fide molecules) but a derived form in the likelihood analyses (fide morphology), largely due to the different relative weighting accorded to data partitions.     

Physiological,Behavioral and Maternal Factors That Contribute to Size Variation in Larval Amphibian Populations

Size variance among similarly aged individuals within populations is a pattern common to many organisms that is a result of interactions between intrinsic and extrinsic traits of individuals. While genetic and maternal effects, as well as physiological and behavioral traits have been shown to contribute to size variation in animal populations, teasing apart the influence of such factors on individual growth rates remain a challenge. Furthermore, tracing the effects of these interactions across life stages and in shaping adult phenotypes also requires further exploration. In this study we investigated the relationship between genetics, hatching patterns, behaviors, neuroendocrine stress axis activity and variance in growth and metamorphosis among same-aged larval amphibians. Through parallel experiments we found that in the absence of conspecific interactions, hatch time and to a lesser extent egg clutch identity (i.e. genetics and maternal effects) influenced the propensity for growth and development in individual tadpoles and determined metamorphic traits. Within experimental groups we found that variance in growth rates was associated with size-dependent foraging behaviors and responses to food restriction. We also found an inverse relationship between glucocorticoid (GC) hormone levels and body mass and developmental stage among group-reared tadpoles, which suggests that GC expression plays a role in regulating differing within-population growth trajectories in response to density-dependent conditions. Taken together these findings suggest that factors that influence hatching conditions can have long-term effects on growth and development. These results also raise compelling questions regarding the extent to which maternal and genetic factors influence physiological and behavioral profiles in amphibians.     

High-frequency monitoring of the genetic diversity and the potential toxicity of a Microcystis aeruginosa bloom in a French shallow lake

During cyanobacterial blooms, processes influencing the population dynamics of blooming species remain partially unexplained. To provide new information, we performed a high-frequency monitoring – every 2 days at six sampling points – of a Microcystis aeruginosa population blooming in a shallow lake. At each sampling date, there was no spatial heterogeneity in the ITS genotypic composition of the population and in the proportion of potentially microcystin- producing (mcyB+) cells, whereas high variations were recorded in cell abundances. In contrast, when looking at the temporal evolution of these parameters, the ITS genotypic composition of the population and in a lesser extent the percentage of mcyB+ cells displayed high variations during the growth phase of the bloom, but not during the plateau phase or the subsequent decline. This suggests that during the development of the bloom, there was no directional selection leading to the dominance of a restricted number of genotypes and that a balancing selection process permitted the maintenance of a high genetic diversity in the Microcystis population. Finally, no relationship was found between these variations occurring in the Microcystis population and those recorded for several environmental parameters, suggesting that many factors and processes interacting together might be involved in these variations.