Cardiomyopathy Mutations in the Tail of β-Cardiac Myosin Modify the Coiled-coil Structure and Affect Integration into Thick Filaments in Muscle Sarcomeres in Adult Cardiomyocytes

It is unclear why mutations in the filament-forming tail of myosin heavy chain (MHC) cause hypertrophic or dilated cardiomyopathy as these mutations should not directly affect contraction. To investigate this, we first investigated the impact of five hypertrophic cardiomyopathy-causing (N1327K, E1356K, R1382W, E1555K, and R1768K) and one dilated cardiomyopathy-causing (R1500W) tail mutations on their ability to incorporate into muscle sarcomeres in vivo. We used adenoviral delivery to express full-length wild type or mutant enhanced GFP-MHC in isolated adult cardiomyocytes. Three mutations (N1327K, E1356K, and E1555K) reduced enhanced GFP-MHC incorporation into muscle sarcomeres, whereas the remainder had no effect. No mutations significantly affected contraction. Fluorescence recovery after photobleaching showed that fluorescence recovery for the mutation that incorporated least well (N1327K) was significantly faster than that of WT with half-times of 25.1 ± 1.8 and 32.2 ± 2.5 min (mean ± S.E.), respectively. Next, we determined the effects of each mutation on the helical properties of wild type and seven mutant peptides (7, 11, or 15 heptads long) from the myosin tail by circular dichroism. R1382W and E1768K slightly increased the α-helical nature of peptides. The remaining mutations reduced α-helical content, with N1327K showing the greatest reduction. Only peptides containing residues 1301–1329 were highly α-helical suggesting that this region helps in initiation of coiled coil. These results suggest that small effects of mutations on helicity translate into a reduced ability to incorporate into sarcomeres, which may elicit compensatory hypertrophy.     

Antimicrobial and antioxidative potential of free and immobilised cellobiose dehydrogenase isolated from wood degrading fungi

Cellobiose dehydrogenase (CDH, EC 1.1.99.18) is a glycoprotein having many biotechnological applications. In the present study, CDHs isolated from Phlebia lindtneri (PlCDH), Phanerochaete chrysosporium (PchCDH), Cerrena unicolor (CuCDH), and Pycnoporus sanguineus (PsCDH) were studied the first time for their ability to generate antioxidant and antimicrobial agents. The aim of the research was to evaluate the antioxidant and antimicrobial activity of systems composed of four CDHs and lactose or cellobiose as a reaction substrate. The free radical scavenging effect of free and immobilised enzymes was evaluated using the DPPH method. The lowest values of EC₅₀ (10.04 ± 0.75 $\text{μg}$/ml) was noted for PlCDH/lactose and for PlCDH/cellobiose (12.06 ± 1.35 $\text{μg}$/ml). The EC₅₀value reached 12.6 ± 1.51 $\text{μg}$/ml in the case of PsCDH/lactose and 15.96 ± 1.35 for PsCDH. The CDH preparations were also effectively immobilised in alginate (the immobilisation efficiency expressed as a protein yield ranged from 61.6 to 100 %). The operational stability expressed as a scavenging effect showed the possibility of using the alginate beads 4 times. Both the free and immobilised CDHs as well as the CDH/substrate were tested against Gram-negative Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Gram-positive Staphylococcus aureus ATCC 25923 bacteria. All samples, except PlCDH, were potentially effective in suppression of bacterial growth. The highest percentage of inhibition (100 %) was obtained for S. aureus bacteria using PsCDH and PchCDH with lactose as a substrate, whereas a slightly lesser effect was observed for E. coli and P. aeruginosa bacterial cells, i.e. 64.1 % and 86.5 % (PsCDH) and 94.1 % and 41.4 % (PchCDH), respectively. Furthermore, the concentrations of the reaction products (aldonic acids and hydrogen peroxide) were quantified and the surface morphology of the alginate beads was analysed using SEM visualisation.     

Substrate moisture,particle size and temperature preferences of trap‐building larvae of sympatric antlions and wormlions from the rainforest of Borneo

1. Larvae of antlions (Neuroptera: Myrmeleontidae) and wormlions (Diptera: Vermileonidae) display a convergently evolved sit‐and‐wait hunting strategy of building pitfall traps in sandy areas. This study investigated a sympatric population of antlions and wormlions in the lowland rainforest of Borneo for substrate moisture, particle size and temperature preferences. It was hypothesised that these animals would show different preferences regarding these microhabitat traits. 2. The results showed that antlions had a higher aversion to moisture compared with wormlions, but that wormlions had a higher preference for small‐particle sand. Furthermore, thermal preferences in antlions and wormlions were significantly different, with antlions choosing higher temperatures. 3. The detected differences between antlions and wormlions might contribute to their niche partitioning in the mixed Bornean population and thus facilitate coexistence of these animals. It is possible that the hotter and dryer microhabitat edges are preferred by antlions.     

Invariant chain processing is independent of cathepsin variation between primary human B cells/dendritic cells and B-lymphoblastoid cells

As part of the endocytic antigen processing pathway, proteolytic cleavage of the invariant chain (Ii) is important for the generation of class II-associated invariant chain peptide (CLIP). CLIP remains associated with the major histocompatibility complex (MHC) class II molecule to prevent premature loading of antigenic peptides. Cysteine proteases, such as Cathepsin S (CatS), CatL, or CatV, play a pivotal role in the final stage of Ii degradation depending on the cell type studied. Less is known regarding the early stages of Ii processing. We therefore explored whether the serine protease CatG is involved in the initial step of Ii degradation in primary antigen presenting cells (APC), since the cathepsin distribution differs between primary APC and cell lines. While primary human B cells and dendritic cells (DC) do harbor CatG, this protease is absent in B-lymphoblastoid cells (BLC) or monocyte-derived DC generated in vitro. In addition, other proteases, such as CatC, CatL, and the asparagine endoprotease (AEP), are active in BLC and monocyte-derived DC. Here we demonstrate that CatG progressively degraded Ii in vitro resulting in several intermediates. However, pharmacological inhibition of CatG in primary B cells and DC did not alter Ii processing, indicating that CatG is dispensable in Ii degradation. Interestingly, stalling of cysteine proteases by inhibition in BLC vs. primary B cells and DC did not result in any differences in the generation of distinct Ii intermediates between the cells tested, suggesting that Ii processing is independent of the cathepsin variation within professional human APC.     

Significant genetic differentiation between Poland and Germany follows present-day political borders, as revealed by Y-chromosome analysis

To test for human population substructure and to investigate human population history we have analysed Y-chromosome diversity using seven microsatellites (Y-STRs) and ten binary markers (Y-SNPs) in samples from eight regionally distributed populations from Poland (n=913) and 11 from Germany (n=1,215). Based on data from both Y-chromosome marker systems, which we found to be highly correlated (r=0.96), and using spatial analysis of the molecular variance (SAMOVA), we revealed statistically significant support for two groups of populations: (1) all Polish populations and (2) all German populations. By means of analysis of the molecular variance (AMOVA) we observed a large and statistically significant proportion of 14% (for Y-SNPs) and 15% (for Y-STRs) of the respective total genetic variation being explained between both countries. The same population differentiation was detected using Monmoniers algorithm, with a resulting genetic border between Poland and Germany that closely resembles the course of the political border between both countries. The observed genetic differentiation was mainly, but not exclusively, due to the frequency distribution of two Y-SNP haplogroups and their associated Y-STR haplotypes: R1a1*, most frequent in Poland, and R1*(xR1a1), most frequent in Germany. We suggest here that the pronounced population differentiation between the two geographically neighbouring countries, Poland and Germany, is the consequence of very recent events in human population history, namely the forced human resettlement of many millions of Germans and Poles during and, especially, shortly after World War II. In addition, our findings have consequences for the forensic application of Y-chromosome markers, strongly supporting the implementation of population substructure into forensic Y chromosome databases, and also for genetic association studies.     

MicroRNA-181a/b-1 Is Not Required for Innate γδ NKT Effector Cell Development

Thymic development of αβ T lymphocytes into invariant natural killer (NK) T cells depends on their selection via agonistic lipid antigen presented by CD1d. If successful, newly selected NKT cells gain effector functions already in the thymus. Some γδ T cell subsets also acquire effector functions in the thymus. However, it is not clear whether agonistic TCR stimulation is involved in thymic γδ T cell selection and development. Here we combine two genetic models to address this question. MiR-181a/b-1^–/–mice, which show impaired agonistic T cell selection of invariant αβ NKT cells, were crossed to Tcrd-H2BeGFP reporter mice to monitor selection, intra-thymic expansion and differentiation of γδ T cells. We found that miR-181a/b-1-deficiency had no effect on numbers of thymic γδ T cell or on their differentiation towards an IL-17- or IFN-γ-producing effector phenotype. Also, the composition of peripheral lymph node γδ T cells was not affected by miR-181a/b-1-deficiency. Dendritic epidermal γδ T cells were normally present in knock-out animals. However, we observed elevated frequencies and numbers of γδ NKT cells in the liver, possibly because γδ NKT cells can expand and replace missing αβ NKT cells in peripheral niches. In summary, we investigated the role of miR-181a/b-1 for selection, intrathymic development and homeostasis of γδ T cells. We conclude that miR-181a/b-1-dependent modulation of T cell selection is not critically required for innate development of γδ NKT cells or of any other γδ T cell subtypes.     

Fine root classification matters: nutrient levels in different functional categories,orders and diameters of roots in boreal Pinus sylvestris across a latitudinal gradient

Plant and Soil - Any grouping of tree species concerned with SOC sequestration should include trees that are as homogeneous as possible in their carbon sequestration. We propose a classification of...     

Inulinolytic activity of broths of Aspergillus niger ATCC 204447 cultivated in shake flasks and stirred tank bioreactor

It is the first detailed study of an inulinolytic fungus Aspergillus niger ATCC 204447 since its discovery, covering submerged cultivations both in shake flasks and a stirred tank bioreactor. Various carbon sources were applied to induce the inulinolytic activity in shake flask cultures. The highest volumetric and specific (per gram of biomass) activities (respectively 0.68 U/mL and 184 U g/X) were observed for the initial inulin and sucrose concentrations equal to 20 g/L. The fungus grew as large (>3 mm) spherical pellets. The influence of inoculum density and application of microparticle‐enhanced cultivation (MPEC) were studied in the batch bioreactor cultivations. Inoculum density moderately affected the inulinolytic activities, whose highest values were 0.7 U/mL and 165 U g/X at the lowest studied spore density of 3.33·10⁸ L^?1. Dispersed hyphae evolved in the bioreactor made the broth difficult to aerate due to high apparent viscosity (exceeding 200 Pa sⁿ at shear rate about 0.05 s^?1) and shear thinned properties (flow behavior index below 0.2). In MPEC (10 μm talc microparticles) the pellets of diameter between 1 and 2 mm were formed, which facilitated the aeration of the broth and increased the specific inulinolytic activity 3.5‐fold.     

A modified neutral comet assay: elimination of lysis at high temperature and validation of the assay with anti-single-stranded DNA antibody

Comet assay under neutral conditions allows the detection of DNA double-strand breaks (DSB), considered to be the biologically relevant radiation-induced lesion. In this report, we describe modifications of the neutral comet method, which simplify and facilitate its use for estimation of DNA DSB in X-irradiated mammalian cells in culture. The analysis carried out according to this protocol takes less time than those most often applied. Also, the use of lysis at 50 degrees C is avoided; this is important in view of the presence of heat-labile sites in the chromatin of irradiated cells, recently reported by Rydberg [Radiation-induced heat-labile sites that convert into DNA double-strand breaks, Radiation Research 153 (2000) 805-812]. The comets have well-defined, sharp limits, suitable for image analysis. The chromatin of the hydrogen peroxide-treated or UV-C-irradiated cell remains condensed similarly to that of the control cells. We checked the neutral comets for the presence of single-stranded DNA by means of a specific antibody. The results point to a satisfactory sensitivity of the modified neutral comet assay and its specificity for DSB. The minimum detection level of the modified neutral comet assay is about 5 Gy.     

Systematic Characterization of Myocardial Inflammation,Repair, and Remodeling in a Mouse Model of Reperfused Myocardial Infarction

Mouse models of myocardial infarction are essential tools for the study of cardiac injury, repair, and remodeling. Our current investigation establishes a systematic approach for quantitative evaluation of the inflammatory and reparative response, cardiac function, and geometry in a mouse model of reperfused myocardial infarction. Reperfused mouse infarcts exhibited marked induction of inflammatory cytokines that peaked after 6 hr of reperfusion. In the infarcted heart, scar contraction and chamber dilation continued for at least 28 days after reperfusion; infarct maturation was associated with marked thinning of the scar, accompanied by volume loss and rapid clearance of cellular elements. Echocardiographic measurements of end-diastolic dimensions correlated well with morphometric assessment of dilative remodeling in perfusion-fixed hearts. Hemodynamic monitoring was used to quantitatively assess systolic and diastolic function; the severity of diastolic dysfunction following myocardial infarction correlated with cardiomyocyte hypertrophy and infarct collagen content. Expression of molecular mediators of inflammation and cellular infiltration needs to be investigated during the first 72 hr, whereas assessment of dilative remodeling requires measurement of geometric parameters for at least four weeks after the acute event. Rapid initiation and resolution of the inflammatory response, accelerated scar maturation, and extensive infarct volume loss are important characteristics of infarct healing in mice.