Effects of population size and forest management on genetic diversity and structure of the tuberous orchid Orchis mascula

Due to societal changes and altered demands for firewood, the traditional forest management of coppicing has been largely abandoned. As a result, many forest herbs that are specifically adapted to regular opening of the canopy, have suffered significant declines in abundance, and the remaining populations of these species often tend to be small and isolated. Reduced population sizes and pronounced spatial isolation may cause loss of within-population genetic diversity and increased between-population differentiation through random genetic drift and inbreeding. In this study, we investigated genetic diversity and genetic structure of 15 populations of the food-deceptive orchid Orchis mascula using AFLP markers. Within-population genetic diversity significantly increased with increasing population size, indicating genetic impoverishment in small populations. Genetic differentiation, on the other hand, was rather low (Φ_ST = 0.083) and there was no significant relationship between genetic and geographic distances, suggesting substantial gene flow within the study area. However, strong differences in levels of within-population diversity and among-population differentiation were found for populations located in forests that have been regularly coppiced and populations found in forests that were neglected for more than 50 years and that were totally overgrown by shrubs. Our data thus indicate that a lack of coppicing leads to decreased genetic diversity and increased differentiation in this orchid species, most likely as a result of genetic drift following demographic bottlenecks. From a conservation point of view, this study combined with previous results on the demography of O. mascula in relation to forest management illustrates the importance of coppicing in maintaining viable populations of forest herbs in the long-term.     

Metabolite Profiling Identifies Candidate Markers Reflecting the Clinical Adaptations Associated with Roux-en-Y Gastric Bypass Surgery

Background

Roux-en-Y gastric bypass (RYGB) surgery is associated with weight loss, improved insulin sensitivity and glucose homeostasis, and a reduction in co-morbidities such as diabetes and coronary heart disease. To generate further insight into the numerous metabolic adaptations associated with RYGB surgery, we profiled serum metabolites before and after gastric bypass surgery and integrated metabolite changes with clinical data.

Methodology and Principal Findings

Serum metabolites were detected by gas and liquid chromatography-coupled mass spectrometry before, and 3 and 6 months after RYGB in morbidly obese female subjects (n = 14; BMI = 46.2±1.7). Subjects showed decreases in weight-related parameters and improvements in insulin sensitivity post surgery. The abundance of 48% (83 of 172) of the measured metabolites changed significantly within the first 3 months post RYGB (p<0.05), including sphingosines, unsaturated fatty acids, and branched chain amino acids. Dividing subjects into obese (n = 9) and obese/diabetic (n = 5) groups identified 8 metabolites that differed consistently at all time points and whose serum levels changed following RYGB: asparagine, lysophosphatidylcholine (C18:2), nervonic (C24:1) acid, p-Cresol sulfate, lactate, lycopene, glucose, and mannose. Changes in the aforementioned metabolites were integrated with clinical data for body mass index (BMI) and estimates for insulin resistance (HOMA-IR). Of these, nervonic acid was significantly and negatively correlated with HOMA-IR (p = 0.001, R = −0.55).

Conclusions

Global metabolite profiling in morbidly obese subjects after RYGB has provided new information regarding the considerable metabolic alterations associated with this surgical procedure. Integrating clinical measurements with metabolomics data is capable of identifying markers that reflect the metabolic adaptations following RYGB.     

The CW domain, a new histone recognition module in chromatin proteins

Post-translational modifications of the N-terminal histone tails, including lysine methylation, have key roles in regulation of chromatin and gene expression. A number of protein modules have been identified that recognize differentially modified histone tails and provide their proteins with the capacity to sense such modifications. Here, we identify the CW domain of plant and animal chromatin-related proteins as a novel module that recognizes different methylated states of lysine 4 on histone H3 (H3K4me). The solution structure of the CW domain of the Arabidopsis ASH1 HOMOLOG2 (ASHH2) histone methyltransferase provides insight into how different CW domains can distinguish different methylated histone tails. We provide evidence that ASHH2 is acting on H3K4me-marked genes, allowing for ASHH2-dependent H3K36 tri-methylation, which contributes to sustained expression of tissue-specific and developmentally regulated genes. This suggests that ASHH2 is a combined 'reader' and 'writer' of the histone code. We propose that different CW domains, dependent on their specificity for different H3K4 methylations, are important for epigenetic memory or participate in switching between permissive and repressive chromatin states.     

HIV-1 Gag extension: conformational changes require simultaneous interaction with membrane and nucleic acid

The retroviral Gag polyprotein mediates viral assembly. The Gag protein has been shown to interact with other Gag proteins, with the viral RNA, and with the cell membrane during the assembly process. Intrinsically disordered regions linking ordered domains make characterization of the protein structure difficult. Through small-angle scattering and molecular modeling, we have previously shown that monomeric human immunodeficiency virus type 1 (HIV-1) Gag protein in solution adopts compact conformations. However, cryo-electron microscopic analysis of immature virions shows that in these particles, HIV-1 Gag protein molecules are rod shaped. These differing results imply that large changes in Gag conformation are possible and may be required for viral formation. By recapitulating key interactions in the assembly process and characterizing the Gag protein using neutron scattering, we have identified interactions capable of reversibly extending the Gag protein. In addition, we demonstrate advanced applications of neutron reflectivity in resolving Gag conformations on a membrane. Several kinds of evidence show that basic residues found on the distal N- and C-terminal domains enable both ends of Gag to bind to either membranes or nucleic acid. These results, together with other published observations, suggest that simultaneous interactions of an HIV-1 Gag molecule with all three components (protein, nucleic acid, and membrane) are required for full extension of the protein.     

On the Role of the SP1 Domain in HIV-1 Particle Assembly: a Molecular Switch?

Expression of a retroviral protein, Gag, in mammalian cells is sufficient for assembly of immature virus-like particles (VLPs). VLP assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We have investigated the role of SP1, a spacer between CA and NC in HIV-1 Gag, in VLP assembly. Mutational analysis showed that even subtle changes in the first 4 residues of SP1 destroy the ability of Gag to assemble correctly, frequently leading to formation of tubes or other misassembled structures rather than proper VLPs. We also studied the conformation of the CA-SP1 junction region in solution, using both molecular dynamics simulations and circular dichroism. Consonant with nuclear magnetic resonance (NMR) studies from other laboratories, we found that SP1 is nearly unstructured in aqueous solution but undergoes a concerted change to an α-helical conformation when the polarity of the environment is reduced by addition of dimethyl sulfoxide (DMSO), trifluoroethanol, or ethanol. Remarkably, such a coil-to-helix transition is also recapitulated in an aqueous medium at high peptide concentrations. The exquisite sensitivity of SP1 to mutational changes and its ability to undergo a concentration-dependent structural transition raise the possibility that SP1 could act as a molecular switch to prime HIV-1 Gag for VLP assembly. We suggest that changes in the local environment of SP1 when Gag oligomerizes on nucleic acid might trigger this switch.     

A New Metabolomic Signature in Type-2 Diabetes Mellitus and Its Pathophysiology

Objective

The objective of the current study was to find a metabolic signature associated with the early manifestations of type-2 diabetes mellitus.

Research Design and Method

Modern metabolic profiling technology (MxP™ Broad Profiling) was applied to find early alterations in the plasma metabolome of type-2 diabetic patients. The results were validated in an independent study. Eicosanoid and single inon monitoring analysis (MxP™ Eicosanoid and MxP™ SIM analysis) were performed in subsets of samples.

Results

A metabolic signature including significantly increased levels of glyoxylate as a potential novel marker for early detection of type-2 diabetes mellitus was identified in an initial study (Study1). The signature was significantly altered in fasted diabetic and pre-diabetic subjects and in non-fasted subjects up to three years prior to the diagnosis of type-2 diabetes; most alterations were also consistently found in an independent patient group (Study 2). In Study 2 diabetic and most control subjects suffered from heart failure. In Study 1 a subgroup of diabetic subjects, with a history of use of anti-hypertensive medication further showed a more pronounced increase of glyoxylate levels, compared to a non-diabetic control group when tested in a hyperglycemic state. In the context of a prior history of anti-hypertensive medication, alterations in hexosamine and eicosanoid levels were also found.

Conclusion

A metabolic signature including glyoxylate was associated with type-2 diabetes mellitus, independent of the fasting status and of occurrence of another major disease. The same signature was also found to be associated with pre-diabetic subjects. Glyoxylate levels further showed a specifically strong increase in a subgroup of diabetic subjects. It could represent a new marker for the detection of medical subgroups of diabetic subjects.     

Microtopography and the Properties of Residual Peat Are Convenient Indicators for Restoration Planning of Abandoned Extracted Peatlands

The natural recovery of vegetation on abandoned peat extraction areas lasts for decades and the result of restoration succession can be unpredictable. The aim of the study was to specify environmental factors that affect the formation of the pioneer stages of mire communities and, therefore, be helpful in the prediction of the resulting ecosystem properties. We used the national inventory data from 64 milled peatlands in Estonia, distributed over the region of 300 × 200 km. This is the first national‐scale statistical evaluation of abandoned extracted peatlands. During surveys, vascular plants, bryophytes, and residual peat properties were recorded on three microtopographic forms: flats, ditch margins, and ditches. The microtopography was the main factor distinguishing the composition of plant communities on flats and ditches, while ditch margins resembled flats. The extracted indicator species suggested two successional pathways, toward fen or raised bog community. A single indicator trait—the depth of residual peat, which combines the information about peat properties (e.g. pH, ash content, and trophicity status), predicted the plant community succession in microtopographic habitats. We suggest that peatland management plans about the cost‐efficient restoration of abandoned peat mining areas should consider properties of residual peat layer as the baseline indicator: milled peatfields with thin (<2.3 m) and well‐decomposed residual peat should be restored toward fen vegetation types, whereas sites with thick (>2.3 m) and less decomposed residual peat layer should be restored toward transitional mires or raised bogs. Specific methodological suggestions are provided .     

Molecular Basis of Calpain Cleavage and Inactivation of the Sodium-Calcium Exchanger 1 in Heart Failure

Cardiac sodium (Na⁺)-calcium (Ca²⁺) exchanger 1 (NCX1) is central to the maintenance of normal Ca²⁺ homeostasis and contraction. Studies indicate that the Ca²⁺-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca²⁺ binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met³⁶⁹ antibody identified a novel calpain cleavage site at Met³⁶⁹. Engineering NCX1-Met³⁶⁹ into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met³⁶⁹ inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met³⁶⁹ cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction.     

Constitutive Nuclear Localization of an Alternatively Spliced Sirtuin-2 Isoform

Sirtuin-2 (SIRT2), the cytoplasmic member of the sirtuin family, has been implicated in the deacetylation of nuclear proteins. Although the enzyme has been reported to be located to the nucleus during G₂/M phase, its spectrum of targets suggests functions in the nucleus throughout the cell cycle. While a nucleocytoplasmic shuttling mechanism has been proposed for SIRT2, recent studies have indicated the presence of a constitutively nuclear isoform. Here we report the identification of a novel splice variant (isoform 5) of SIRT2 that lacks a nuclear export signal and encodes a predominantly nuclear isoform. This novel isoform 5 fails to show deacetylase activity using several assays, both in vitro and in vivo, and we are led to conclude that this isoform is catalytically inactive. Nevertheless, it retains the ability to interact with p300, a known interaction partner. Moreover, changes in intrinsic tryptophan fluorescence upon denaturation indicate that the protein is properly folded. These data, together with computational analyses, confirm the structural integrity of the catalytic domain. Our results suggest an activity-independent nuclear function of the novel isoform.     

The role of soil conditions in fine root ecomorphology in Norway spruce (Picea abies (L.) Karst.)

The present study is an attempt to investigate the pattern of morphological variability of the short roots of Norway spruce (Picea abies (L.) Karst.) growing in different soils. Five root parameters – diameter, length and dry weight of the root tip, root density (dry weight per water-saturated volume) and specific root area (absorbing area of dry weight unit) were studied with respect to 11 soil characteristics using CANOCO RDA analysis. The investigation was conducted in seven study areas in Estonia differing in site quality class and soil type. Ten root samples per study area were collected randomly from the forest floor and from the 20 cm soil surface layer. Eleven soil parameters were included in the study: humus content, specific soil surface area, field capacity, soil bulk density, pH (KCl and H₂O dilution's), N and Ca concentrations, Ca/Al and C/N ratios, and the decomposition rate of fine roots (<2 mm dia.). Root morphological characteristics most strongly related to the measured soil characteristics in the different sites were specific root area, root density and diameter of the short roots, the means varying from 29 to 42 m² kg⁻¹, from 310 to 540 kg m⁻³ and from 0.26 to 0.32 mm, respectively; root density being most sensitive. The most favourable site and soil types resulting in fine roots with morphological characteristics for optimizing nutrient uptake (e.g. low short root density and high specific root area) were Umbric Luvisol (Oxalis), Dystric Gleysol (Oxalis) and Gleyic Luvisol (Hepatica). These soil types correspond to highly productive natural forest stands of Norway spruce in Estonia. All measured soil variables explained 28% of total variance of the root characteristics. The most important variables related to root morphology were the humus content, field capacity and specific soil surface area. This revised version was published online in June 2006 with corrections to the Cover Date.