Global DNA promoter methylation in frontal cortex of alcoholics and controls

To determine if ethanol consumption and alcoholism cause global DNA methylation disturbances, we examined alcoholics and controls using methylation specific microarrays to detect all annotated gene and non-coding microRNA promoters and their CpG islands. DNA was isolated and immunoprecipitated from the frontal cortex of 10 alcoholics and 10 age and gender-matched controls then labeled prior to co-hybridization. A modified Kolmogorov–Smirnov test was used to predict differentially enriched regions (peaks) from log-ratio estimates of amplified vs input DNA. More than 180,000 targets were identified for each subject which correlated with > 30,000 distinct, integrated peaks or high probability methylation loci. Peaks were mapped to regions near 17,810 separate annotated genes per subject representing hypothetical methylation targets. No global methylation differences were observed between the two subject groups with 80% genetic overlap, but extreme methylation was observed in both groups at specific loci corresponding with known methylated genes (e.g., H19) and potentially other genes of unknown methylation status. Methylation density patterns targeting CpG islands visually correlated with recognized chromosome banding. Our study provides insight into global epigenetic regulation in the human brain in relationship to controls and potentially novel targets for hypothesis generation and follow-up studies of alcoholism.     

Diversity patterns of plant functional types in relation to fire regime and previous land use in Mediterranean woodlands

Abstract. We explore patterns of diversity of plant functional types (PFTs) in Mediterranean communities subjected to landscape‐scale fire disturbances in a mosaic of uncultivated and old fields stands. We use regenerative and growth form attributes to establish two sets of PFTs of perennial species living in shrublands and pine forests of NE Spain. We test the following hypotheses: 1. Fire frequency decreases regenerative PFTs diversity by negatively selecting attributes with low regenerative efficiency. 2. Fire history has more influence on regenerative than on growth form PFTs. 3. The lowest diversity of growth form PFTs will be in old fields without recent fires. We surveyed stands of different combinations of fire and land use histories. Fire history included areas without fires in the last twenty years (unburned), sites burned in 1982 (1‐burned), and sites burned in 1982 and 1994 (2‐burned). Land use histories considered terraced old fields, and uncultivated stands on stony soils. We analysed patterns of PFT abundance and diversity at the stand level, and across the landscape (among stands absolute deviations from sample medians of the relative cover of PFTs). At the stand level, fire had more influence on the diversity patterns of regenerative PFTs than on growth form PFTs. Fire decreased the diversity of regenerative PFTs, due to the elimination of the species without effective mechanisms to post‐fire regeneration. This effect was not observed across the landscape, but seeders showed more variation in stands with longer history without fire. Land use contributed to explain the diversity patterns of growth form PFT (i.e. the number of growth form PFTs was lower in uncultivated, unburned sites), but it did not influence regenerative PFTs diversity. Patterns of PFTs diversity reflect the response to ecological processes operating at the landscape level. Overall, regenerative and growth form PFTs appear to be more sensitive to the fire history than to the past land use.     

Effects of fire severity in a north Patagonian subalpine forest

Abstract. Question: What is the relative importance of fire‐induced canopy mortality, soil burning and post‐fire herbivory on tree seedling performance? Location: Subalpine Nothofagus pumilio forests at Challhuaco valley (41°13'S, 71°19'W), Nahuel Huapi National Park, Argentina. Methods: We fenced and transplanted soils of three burning severities along a fire severity gradient produced by a fire in 1996. Over two growing seasons we monitored soil water, direct incoming solar radiation, seedling survival, final seedling total biomass and root/shoot ratio. Additionally, we assessed severity‐related changes in soil properties. Results: Incoming radiation (an indicator of the amount of canopy cover left by the fire) was the primary factor influencing spring and summer top soil water availability, first and second‐year seedling survival and seedling growth. While seedling survival and soil water content were negatively affected by increased radiation, seedling final biomass was highest in very open microsites. Burned soils showed lower water holding capacity and soil carbon; however these changes did not affect topsoil water, and, contrary to expectation, there was a slight tendency toward higher seedling survival on more heavily burned soils. Herbivory significantly reduced seedling survival, but only under high‐radiation conditions. While the effect of radiation on final seedling biomass was not affected by herbivory, R/S ratios were significantly reduced by herbivory in high radiation micro sites. Conclusions: Despite inducing faster aerial growth, increased radiation and herbivory in severely burned sites may effectively prevent post‐fire regeneration in north Patagonian subalpine forest where seed sources are not limiting.     

Post-translational modification of cellular proteins during Leishmania donovani differentiation

The pathogenic intracellular parasites Leishmania donovani cycle between sand fly gut and the human macrophage phagolysosome, differentiating from extracellular promastigotes to intracellular amastigote forms. Using isobaric tagging for relative and absolute quantifications (iTRAQ/LC-MS/MS) proteomic methodology, we recently described the ordered gene expression changes during this process. While protein abundance changes in Leishmania were documented, little is known about their PTMs. Here we used iTRAQ to detect protein phosphorylation, methylation, acetylation, and glycosylation sites throughout differentiation. We found methylation of arginines, aspartic acids, glutamic acids, asparagines, and histidines. Detected acetylation sites included serines and protein N-terminal acetylations on methionines, serines, alanines, and threonines. Phosphorylations were detected on serines and threonines, but not tyrosines. iTRAQ identified novel fucosylation sites as well as hexosylations. We observed quantity changes in some modifications during differentiation, suggesting a role in L. donovani intracellular development. This study is the first high-throughput analysis of PTM sites dynamics during an intracellular parasitic development.     

Involvement of post-translational modification of neuronal plasticity-related proteins in hyperalgesia revealed by a proteomic analysis

To clarify roles of an endogenous pain modulatory system of the central nervous system (CNS) in hyperalgesia, we tried to identify qualitative and quantitative protein changes by a proteomic analysis using an animal model of hyperalgesia. Specifically, we first induced functional hyperalgesia on male Wistar rats by repeated cold stress (specific alternation of rhythm in temperature, SART). We then compared proteomes of multiple regions of CNS and the dorsal root ganglion between the hyperalgetic rats and non-treated ones by 2-D PAGE in the pI range of 4.0-7.0. We found that SART changed the proteomes prominently in the mesencephalon and cerebellum. We thus analyzed the two brain regions in more detail using gels with narrower pI ranges. As a result, 29 and 23 protein spots were significantly changed in the mesencephalon and the cerebellum, respectively. We successfully identified 12 protein spots by a MALDI-TOF/TOF MS and subsequent protein database searching. They included unc-18 protein homolog 67K, collapsin response mediator protein (CRMP)-2 and CRMP-4, which were reported to be involved in neurotransmitter release or axon elongation. Interestingly, mRNA expression levels of these three proteins were not changed significantly by the induction of hyperalgesia. Instead, we found that the detected changes in the protein spots are caused by the post-translational modification (PTM) of proteolysis or phosphorylation. Taken together, development of the hyperalgesia would be linked to PTM of these three CNS proteins. PTM regulation may be one of the useful ways to treat hyperalgesia.     

A simple and highly successful C-terminal sequence analysis of proteins by mass spectrometry

A simple and efficient method for C-terminal sequencing of proteins has long been pursued because it would provide substantial information for identifying the covalent structure, including post-translational modifications. However, there are still significant impediments to both direct sequencing from C termini of proteins and specific isolation of C-terminal peptides from proteins. We describe here a highly successful, de novo C-terminal sequencing method of proteins by employing succinimidyloxycarbonylmethyl tris (2,4,6-trimethoxyphenyl) phosphonium bromide and mass spectrometry.     

Gametic interactions promote inbreeding avoidance in house mice

Reproduction among related individuals is generally maladaptive. Inbreeding imposes significant costs on individual reproductive success, and can decrease population fitness. Theory predicts that polyandrous females can avoid inbreeding by exploiting paternity‐biasing mechanisms that enable differential sperm ‘use’. Evidence of sperm selection is difficult to demonstrate because patterns of non‐random paternity can be generated by a variety of different mechanisms. Here, using in vitro fertilisation in mice, we provide evidence of sperm selection at the gametic level. We mixed the sperm of sibling and non‐sibling males, and observed a fertilisation bias towards the sperm of non‐sibling males. The number of motile sperm and sperm swimming performance did not differ between competitors among the replicate assays. Therefore, our result can only be ascribed to egg‐driven sperm selection against related sperm. We conclude that the expression or secretion of gametic proteins could provide the molecular basis for this mechanism of cryptic female choice.