A Hypomorphic Lsd1 Allele Results in Heart Development Defects in Mice

Lysine-specific demethylase 1 (Lsd1/Aof2/Kdm1a), the first enzyme with specific lysine demethylase activity to be described, demethylates histone and non-histone proteins and is essential for mouse embryogenesis. Lsd1 interacts with numerous proteins through several different domains, most notably the tower domain, an extended helical structure that protrudes from the core of the protein. While there is evidence that Lsd1-interacting proteins regulate the activity and specificity of Lsd1, the significance and roles of such interactions in developmental processes remain largely unknown. Here we describe a hypomorphic Lsd1 allele that contains two point mutations in the tower domain, resulting in a protein with reduced interaction with known binding partners and decreased enzymatic activity. Mice homozygous for this allele die perinatally due to heart defects, with the majority of animals suffering from ventricular septal defects. Molecular analyses revealed hyperphosphorylation of E-cadherin in the hearts of mutant animals. These results identify a previously unknown role for Lsd1 in heart development, perhaps partly through the control of E-cadherin phosphorylation.     

Anaerobic Co-Culture of Mesenchymal Stem Cells and Anaerobic Pathogens - A New In Vitro Model System

Background

Human mesenchymal stem cells (hMSCs) are multipotent by nature and are originally isolated from bone marrow. In light of a future application of hMSCs in the oral cavity, a body compartment with varying oxygen partial pressures and an omnipresence of different bacterial species i.e. periodontitis pathogens, we performed this study to gain information about the behavior of hMSC in an anaerobic system and the response in interaction with oral bacterial pathogens.

Methodology/Principal Findings

We established a model system with oral pathogenic bacterial species and eukaryotic cells cultured in anaerobic conditions. The facultative anaerobe bacteria Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were studied. Their effects on hMSCs and primary as well as permanent gingival epithelial cells (Ca9-22, HGPEC) were comparatively analyzed. We show that hMSCs cope with anoxic conditions, since 40% vital cells remain after 72 h of anaerobic culture. The Ca9-22 and HGPEC cells are significantly more sensitive to lack of oxygen. All bacterial species reveal a comparatively low adherence to and internalization into hMSCs (0.2% and 0.01% of the initial inoculum, respectively). In comparison, the Ca9-22 and HGPEC cells present better targets for bacterial adherence and internalization. The production of the pro-inflammatory chemokine IL-8 is higher in both gingival epithelial cell lines compared to hMSCs and Fusobacterium nucleatum induce a time-dependent cytokine secretion in both cell lines. Porphyromonas gingivalis is less effective in stimulating secretion of IL-8 in the co-cultivation experiments.

Conclusions/significance

HMSCs are suitable for use in anoxic regions of the oral cavity. The interaction with local pathogenic bacteria does not result in massive pro-inflammatory cytokine responses. The test system established in this study allowed further investigation of parameters prior to set up of oral hMSC in vivo studies.     

Pharmacological postconditioning by bolus injection of phosphodiesterase-5 inhibitors vardenafil and sildenafil

Postconditioning enables cardioprotection against ischemia/reperfusion injury either by application of short, repetitive ischemic periods or by pharmacological intervention prior to reperfusion. Pharmacological postconditioning has been described for phosphodiesterase-5 inhibitors when the substances were applied as a permanent infusion. For clinical purposes, application of a bolus is more convenient. In a rat heart in situ model of ischemia reperfusion vardenafil or sildenafil were applied as a bolus prior to reperfusion. Cardioprotective effects were found over a broad dosage range. In accordance with current hypotheses on pharmacological postconditioning signaling, the protective effect was mediated by extracellular signal-regulated kinase and protein kinase C pathway. Interestingly, the extent of protection was independent of the concentration applied for both substances. Full protection comparable to ischemic postconditioning was reached with half-maximal human equivalence dose. In contrast, mean arterial pressure dropped upon bolus application in a dose-dependent manner. Taken together, the current study extends previous findings obtained in a permanent infusion model to bolus application. This is an important step toward clinical application of pharmacological postconditioning with sildenafil and vardenafil, especially because the beneficial effects were proven for concentrations with reduced hemodynamic side effects compared to the dosage applied for erectile dysfunction treatment.     

Homooligomeric dA·dU and dA·dT Sequences in Parallel and Antiparallel Strand Orientation: Consequence of the 5-methyl Groups on Stability,Structure and Interaction with the Minor Groove Binding Drug HOECHST 33258

Abstract

Oligodeoxyribonucleotides containing dA·dU base combinations were shown to form parallel stranded DNA. CD spectra and hyperchromicity profiles provide evidence that the structure is very similar to that of a related parallel stranded dA·oligomer. Thermal denaturation studies show that these parallel dAdU sequences are significantly less stable than their dA·analogues in either antiparallel or parallel stranded orientations. The stabilizing effect of the 5- methyl group is similar for parallel and antiparallel sequences. The minor groove binding drug Hoechst 33258 binds with similar affinity to APS dA·and APS dA·dU sequences. However, binding to the PS dA·hairpin is significantly impaired as a consequence of the different groove dimensions and the presence of thymine methyl groups at the binding site. This results in an 8.6 kJmoF reduced free energy of binding for the PS dA·sequence. Replacement of the bulky methyl group with a hydrogen (ie. T -> U) results in significantly stronger Hoechst 33258 binding to the parallel dA·dU sequences with a penalty of only 4.1 kJmol^?1. Our data demonstrate that although Hoechst 33258 detects the altered groove, it is still able to bind a PS duplex containing dA·dU base pairs with high affinity, despite the large structural differences from its regular binding site in APS DNA.     

Towards an Understanding of DNA Recognition by the Methyl-CpG Binding Domain 1

Abstract

CpG methylation determines a variety of biological functions of DNA. The methylation signal is interpreted by proteins containing a methyl-CpG binding domain (MBDs). Based on the NMR structure of MBD1 complexed with methylated DNA we analysed the recognition mode by means of molecular dynamics simulations.

As the protein is monomeric and recognizes a symmetrically methylated CpG step, the recognition mode is an asymmetric one. We find that the two methyl groups do not contribute equally to the binding energy. One methyl group is associated with the major part of the binding energy and the other one nearly does not contribute at all. The contribution of the two cytosine methyl groups to binding energy is calculated to be ?3.6 kcal/mol. This implies a contribution of greater than two orders of magnitude to the binding constant. The conserved amino acid Asp32 is known to be essential for DNA binding by MBD1, but so far no direct contact with DNA has been observed. We detected a direct DNA base contact to Asp32. This could be the main reason for the importance of this amino acid. MBD contacts DNA exclusively in the major groove, the minor groove is reserved for histone contacts. We found a deformation of the minor groove shape due to complexation by MBD1, which indicates an information transfer between the major and the minor groove.     

Genome wide sequence analysis grants unbiased definition of species boundaries in “Candidatus Phytoplasma”

The phytoplasmas are currently named using the Candidatus category, as the inability to grow them in vitro prevented (i) the performance of tests, such as DNA-DNA hybridization, that are regarded as necessary to establish species boundaries, and (ii) the deposition of type strains in culture collections. The recent accession to complete or nearly complete genome sequence information disclosed the opportunity to apply to the uncultivable phytoplasmas the same taxonomic approaches used for other bacteria. In this work, the genomes of 14 strains, belonging to the 16SrI, 16SrIII, 16SrV and 16SrX groups, including the species “Ca. P. asteris”, “Ca. P. mali”, “Ca. P. pyri”, “Ca. P. pruni”, and “Ca. P. australiense” were analyzed along with Acholeplasma laidlawi, to determine their taxonomic relatedness. Average nucleotide index (ANIm), tetranucleotide signature frequency correlation index (Tetra), and multilocus sequence analysis of 107 shared genes using both phylogenetic inference of concatenated (DNA and amino acid) sequences and consensus networks, were carried out. The results were in large agreement with the previously established 16S rDNA based classification schemes. Moreover, the taxonomic relationships within the 16SrI, 16SrIII and 16SrX groups, that represent clusters of strains whose relatedness could not be determined by 16SrDNA analysis, could be comparatively evaluated with non-subjective criteria. “Ca. P. mali” and “Ca. P. pyri” were found to meet the genome characteristics for the retention into two different, yet strictly related species; representatives of subgroups 16SrI-A and 16SrI-B were also found to meet the standards used in other bacteria to distinguish separate species; the genomes of the strains belonging to 16SrIII were found more closely related, suggesting that their subdivision into Candidatus species should be approached with caution.     

Minor contribution of leaf litter to N nutrition of beech (Fagus sylvatica) seedlings in a mountainous beech forest of Southern Germany

Aims

Our aims were to characterize the fate of leaf-litter-derived nitrogen in the plant-soil-microbe system of a temperate beech forest of Southern Germany and to identify its importance for N nutrition of beech seedlings.

Methods

¹⁵N-labelled leaf litter was traced in situ into abiotic and biotic N pools in mineral soil as well as into beech seedlings and mycorrhizal root tips over three growing seasons.

Results

There was a rapid transfer of ¹⁵N into the mineral soil already 21 days after tracer application with soil microbial biomass initially representing the dominant litter-N sink. However, ¹⁵N recovery in non-extractable soil N pools strongly increased over time and subsequently became the dominant ¹⁵N sink. Recovery in plant biomass accounted for only 0.025 % of ¹⁵N excess after 876 days. After three growing seasons, ¹⁵N excess recovery was characterized by the following sequence: non-extractable soil N?>>?extractable soil N including microbial biomass?>>?plant biomass?>?ectomycorrhizal root tips.

Conclusions

After quick vertical dislocation and cycling through microbial N pools, there was a rapid stabilization of leaf-litter-derived N in non-extractable N pools of the mineral soil. Very low ¹⁵N recovery in beech seedlings suggests a high importance of other N sources such as root litter for N nutrition of beech understorey.     

Root system development of Lotus corniculatus L. in calcareous sands with embedded finer-textured fragments in an initial soil

Background and aims

In post mining landscapes as in the Lusatian region (Brandenburg, Germany), Pleistocene coarse-textured, sandy sediments are used for soil rehabilitation and land reclamation. The homogeneously-appearing initial soils are characterized by finer-textured soil clumps (fragments) of different sizes that are embedded in a sandy matrix. These soils with typical local-scale heterogeneity may serve as a model for studying how spatially-distributed soil fragments may be utilized by pioneering plant species. The aim of this study was to gain insight into the physical and chemical properties of sandy matrix and fragments that could possibly explain why embedded fragment may act as hot spots for root growth.

Methods

In 2009, three soil monoliths of dimension 50 cm?×?50 cm?×?50 cm that were exclusively vegetated by Lotus corniculatus L. planted in 2008 were studied. Each layer of 10 cm was sampled successively using a cubic metal frame with 10 cm edge length (25 samples per layer each with a volume of 1 l). The samples were analyzed for root biomass, root lengths and diameter, and for chemical and physical properties of sandy matrix and fragments.

Results

Bulk density, water contents, total carbon, total nitrogen, and plant available calcium contents were higher for the fragments compared to the sandy matrix. The roots of L. corniculatus were heterogeneously distributed in the monoliths. The root density distributions for the 1 L samples indicated a positive influence of fragments on directed root growth. Fragments embedded in the sandy matrix were found to be strongly penetrated by roots despite their relatively high bulk density. The presence of fragments also led to an increased root biomass in the sandy matrix in the direct vicinity of fragments. Such direct effects on root development were accompanied by more indirect effects by locally-elevated moisture and nutrient contents.

Conclusion

The results suggest that finer-textured fragments embedded in coarser-textured sediments, can have favorable effect on plant and root development during the initial stages of establishment of vegetation cover. The fragments can act as water and nutrient hot spots to improve supply of pioneering plants especially in coarse-textured soil. The existence of small-scale heterogeneities owing to incomplete sediment mixing e.g., in soil reclamation, could be generally important for controlling the speed and direction of early plants-establishment, for instance, in the succession of post-mining areas.     

Preferential use of root litter compared to leaf litter by beech seedlings and soil microorganisms

Background and aims

Litter decomposition is regulated by e.g. substrate quality and environmental factors, particularly water availability. The partitioning of nutrients released from litter between vegetation and soil microorganisms may, therefore, be affected by changing climate. This study aimed to elucidate the impact of litter type and drought on the fate of litter-derived N in beech seedlings and soil microbes.

Methods

We quantified ¹⁵N recovery rates in plant and soil N pools by adding ¹⁵N-labelled leaf and/or root litter under controlled conditions.

Results

Root litter was favoured over leaf litter for N acquisition by beech seedlings and soil microorganisms. Drought reduced ¹⁵N recovery from litter in seedlings thereby affecting root N nutrition. ¹⁵N accumulated in seedlings in different sinks depending on litter type.

Conclusions

Root turnover appears to influence (a) N availability in the soil for plants and soil microbes and (b) N acquisition and retention despite a presumably extremely dynamic turnover of microbial biomass. Compared to soil microorganisms, beech seedlings represent a very minor short-term N sink, despite a potentially high N residence time. Furthermore, soil microbes constitute a significant N pool that can be released in the long term and, thus, may become available for N nutrition of plants.