Determination of the DNA methylation level of the marbled crayfish: an increase in sample throughput by an optimised sample preparation

Using a previously described capillary electrophoretic method with laser-induced fluorescence detection the genomic methylation level can be determined exactly. We present a sample preparation that eliminates the surplus of fluorescence marker used for coupling resulting in an increase of sample throughput from 75 to 250 analyses per week. The sensitivity of the method was also increased, which allows the determination of methylation levels under 1%. With these changes in sample preparation a methylation level of 1.64+/-0.03% in hepatopancreas DNA of the recently discovered marbled crayfish could be determined.     

<Emphasis Type="Italic">myo</Emphasis>-Inositol Phosphate Isomers Generated by the Action of a Phytase from a Malaysian Waste-water Bacterium

Using a combination of High-Performance Ion Chromatography analysis and kinetic studies, the pathway of myo-inositol hexakisphosphate dephosphorylation by a phytase from a Malaysian waste-water bacterium was established. The data demonstrate that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-I(1,2,3,4,5)P₅, D-I(2,3,4,5)P₄, D-I(2,3,4)P₃, D-I(2,3)P₂ to finally I(2)P. It was estimated that more than 90% of phytate hydrolysis occurs via D-I(1,2,3,4,5)P₅. Thus, the phytase from the Malaysian waste-water bacterium has to be considered a 6-phytase (E.C. 3.1.3.26). A second pathway of minor importance could be proposed which is in accordance with the results obtained from analysis of the dephosphorylation products formed by the action of the phytase under investigation on myo-inositol hexakisphosphate. It proceeds via D/L-I(1,2,4,5,6)P₅, D/L-I(1,2,4,5)P₄, D/L-I(1,2,4)P₃, D/L-I(2,4)P₂ to finally I(2)P.     

Peptaibol production by sepedonium strains parasitizing boletales

Fungi of the genus Sepedonium (anamorphic ascomycetes) are known to infect fruiting bodies of Basidiomycetes of the order Boletales. We have characterized twelve Sepedonium isolates by intact-cell mass spectrometry (IC-MS) with the help of respective biomarkers and their metabolite spectra focusing on peptaibol production. A strain of mycoparasitic S. chalcipori was grown in solid-state fermentation, and tylopeptin production was found, suggesting an ascomycete origin of these peptaibols, which were first described in the basidiomycete Tylopilus neofelleus. In addition, the structures of two new peptaibols, chalciporin A (=Ac-Trp-Val-Aib-Val-Ala-Gln-Ala-Aib-Ser-Leu-Ala-Leu-Aib-Gln-Leuol) and chalciporin B (=Ac-Trp-Val-Aib-Val-Ala-Gln-Ala-Aib-Gln-Aib-Ala-Leu-Aib-Gln-Leuol) are presented. The IC-MS technique was applied for in situ peptaibol analysis of Sepedonium strains growing on Boletales, in particular S. chrysospermum infecting Xerocomus cf. badius. We found chrysospermins at the surface and within basidiomycete tissue, as well as in the cultivated parasite.     

Modulation of Ca2+ entry and plasma membrane potential by human TRPM4b

TRPM4b is a Ca(2+)-activated, voltage-dependent monovalent cation channel that has been shown to act as a negative regulator of Ca(2+) entry and to be involved in the generation of oscillations of Ca(2+) influx in Jurkat T-lymphocytes. Transient overexpression of TRPM4b as an enhanced green fluorescence fusion protein in human embryonic kidney (HEK) cells resulted in its localization in the plasma membrane, as demonstrated by confocal fluorescence microscopy. The functionality and plasma membrane localization of overexpressed TRPM4b was confirmed by induction of Ca(2+)-dependent inward and outward currents in whole cell patch clamp recordings. HEK-293 cells stably overexpressing TRPM4b showed higher ionomycin-activated Ca(2+) influx than wild-type cells. In addition, analysis of the membrane potential using the potentiometric dye bis-(1,3-dibutylbarbituric acid)-trimethine oxonol and by current clamp experiments in the perforated patch configuration revealed a faster initial depolarization after activation of Ca(2+) entry with ionomycin. Furthermore, TRPM4b expression facilitated repolarization and thereby enhanced sustained Ca(2+) influx. In conclusion, in cells with a small negative membrane potential, such as HEK-293 cells, TRPM4b acts as a positive regulator of Ca(2+) entry.     

Time processing in Huntington's disease: a group-control study

Background

“Timing” processes are mediated via a disturbed neuronal network including the basal ganglia. Brain structures important for “timing” are also discussed to be critical for the deterioration of movements in Huntington''s disease (HD). Changes in “timing processes” are found in HD, but no study has varied the degree of motor demands in timing functions in parallel in HD. It may be hypothesized that timing functions may be deteriorated to a different extent in motor and non-motor timing, because in motor timing the underlying brain structures may be more demanding than in non-motor timing.

Methodology/Principle Findings

We assessed timing in two different experiments: a time-estimation (TE) and a time-discrimination (TD) task. The demand on motor functions is high in the TE-task and low in the TD-task. Furthermore, general motor ability was assessed at different complexity levels. A presymptomatic (pHD), a symptomatic (HD) and a control group were investigated. We found a decline in timing functions when demands on the motor system were high (TE-task), in HD and even in pHD, compared to controls. In non-motor timing (TD task) and in the assessment of general motor ability, performance in the pHD-group was comparable to the controls and better than in the symptomatic group. Performance in both timing tasks was related to the duration until the estimated age of onset in pHDs.

Conclusions/Significance

The study shows a selective deterioration of time-estimation processes in symptomatic and even presymptomatic Huntington''s disease. Time-discrimination processes were not affected in both patient groups. The relation of timing performance to the duration until the estimated age of onset in pHD is of clinical importance.     

Phylogeny of the nematode genus <Emphasis Type="Italic">Pristionchus</Emphasis> and implications for biodiversity,biogeography and the evolution of hermaphroditism

Background  

The nematode Pristionchus pacificus has originally been developed as a satellite organism for comparison to Caenorhabditis elegans. A 10X coverage of the whole genome of P. pacificus is available, making P. pacificus the first non- Caenorhabditis nematode with a fully sequenced genome. The macroevolutionary comparison between P. pacificus and C. elegans has been complemented by microevolutionary studies of closely related strains and species within the genus Pristionchus. In addition, new understanding of the biology of Pristionchus from field studies, demonstrating a close association with various scarab beetles and the Colorado potato beetle, supports consideration of this nematode in studies of ecosystems. In the course of field studies on four continents more than 1,200 isolates were established from 15,000 beetle specimens representing 18 Pristionchus species. Two remarkable features of the Pristionchus – beetle association are the high species specificity of the interaction and the interception of the beetle's sex communication system for host recognition by the nematodes, as suggested by chemotaxis studies. Evolutionary interpretations of differences in developmental, behavioral and ecological patterns require a phylogenetic framework of the genus Pristionchus.     

Quaternary structure and apical membrane sorting of the mammalian NaSi-1 sulfate transporter in renal cell lines

NaSi-1 encodes a Na⁺-sulfate cotransporter expressed on the apical membrane of renal proximal tubular cells, which is responsible for body sulfate homeostasis. Limited information is available on NaSi-1 protein structure and the mechanisms controlling its apical membrane sorting. The aims of this study were to biochemically determine the quaternary structure of the rat NaSi-1 protein and to characterize its expression in renal epithelial cell lines. Hexahistidyl-tagged NaSi-1 (NaSi-1-His) proteins expressed in Xenopus oocytes, appeared as two bands of about 60 and 75 kDa. PNGase F treatment shifted both bands to 57 kDa while endoglycosidase H treatment led to a downward shift of the lower molecular mass band only. Mutagenesis of a putative N-glycosylation site (N591S) produced a single band that was not shifted by endoglycosidase H or PNGase F, confirming a single glycosylation site at residue 591. Blue native-PAGE and cross-linking experiments revealed dimeric complexes, suggesting the native form of NaSi-1 to be a dimer. Transient transfection of EGFP/NaSi-1 in renal epithelial cells (OK, LLC-PK1 and MDCK) demonstrated apical membrane sorting, which was insensitive to tunicamycin. Transfection of the EGFP/NaSi-1 N591S glycosylation mutant also showed apical expression, suggesting N591 is not essential for apical sorting. Treatment with cholesterol depleting compounds did not disrupt apical sorting, but brefeldin A led to misrouting to the basolateral membrane, suggesting that NaSi-1 sorting is through the ER to Golgi pathway. Our data demonstrates that NaSi-1 forms a dimeric protein which is glycosylated at N591, whose sorting to the apical membrane in renal epithelial cells is brefeldin A-sensitive and independent of lipid rafts or glycosylation.     

Vorolign--fast structural alignment using Voronoi contacts

Vorolign, a fast and flexible structural alignment method for two or more protein structures is introduced. The method aligns protein structures using double dynamic programming and measures the similarity of two residues based on the evolutionary conservation of their corresponding Voronoi-contacts in the protein structure. This similarity function allows aligning protein structures even in cases where structural flexibilities exist. Multiple structural alignments are generated from a set of pairwise alignments using a consistency-based, progressive multiple alignment strategy. RESULTS: The performance of Vorolign is evaluated for different applications of protein structure comparison, including automatic family detection as well as pairwise and multiple structure alignment. Vorolign accurately detects the correct family, superfamily or fold of a protein with respect to the SCOP classification on a set of difficult target structures. A scan against a database of >4000 proteins takes on average 1 min per target. The performance of Vorolign in calculating pairwise and multiple alignments is found to be comparable with other pairwise and multiple protein structure alignment methods. AVAILABILITY: Vorolign is freely available for academic users as a web server at http://www.bio.ifi.lmu.de/Vorolign     

SSU rRNA reveals a sequential increase in shell complexity among the euglyphid testate amoebae (Rhizaria: Euglyphida)

The existing data on the molecular phylogeny of filose testate amoebae from order Euglyphida has revealed contradictions between traditional morphological classification and SSU rRNA phylogeny and, moreover, the position of several important genera remained unknown. We therefore carried out a study aiming to fill several important gaps and better understand the relationships among the main euglyphid testate amoebae and the evolutionary steps that led to the present diversity at a higher level. We obtained new SSU rRNA sequences from five genera and seven species. This new phylogeny obtained shows that (1) the clade formed by species of genera Assulina and Placocista branches unambiguously at the base of the subclade of Euglyphida comprising all members of the family Trinematidae and genus Euglypha, (2) family Trinematidae (Trachelocorythion, Trinema, and Corythion) branches as a sister group to genus Euglypha, (3) three newly sequenced Euglypha species (E. cf. ciliata, E. penardi, and E. compressa) form a new clade within the genus. Since our results show that Assulina and Placocista do not belong to the Euglyphidae (unless the Trinematidae are also included in this family), we propose the creation of a new family named Assulinidae. Consequently, we give a family status to the genera Euglypha and (tentatively) Scutiglypha, which become the new family Euglyphidae. The evolutionary pattern suggested by SSU rRNA phylogeny shows a clear tendency towards increasing morphological complexity of the shell characterised by changes in the symmetry (migration of the aperture to a ventral position and/or compression of the shell) and the appearance of specialised scales at the aperture (in families Trinematidae and Euglyphidae).     

Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins

Non-structural protein 1 (NS1) is one of the most enigmatic proteins of the Dengue virus (DENV), playing distinct functions in immune evasion, pathogenesis and viral replication. The recently reported crystal structure of DENV NS1 revealed its peculiar three-dimensional fold; however, detailed information on NS1 function at different steps of the viral replication cycle is still missing. By using the recently reported crystal structure, as well as amino acid sequence conservation, as a guide for a comprehensive site-directed mutagenesis study, we discovered that in addition to being essential for RNA replication, DENV NS1 is also critically required for the production of infectious virus particles. Taking advantage of a trans-complementation approach based on fully functional epitope-tagged NS1 variants, we identified previously unreported interactions between NS1 and the structural proteins Envelope (E) and precursor Membrane (prM). Interestingly, coimmunoprecipitation revealed an additional association with capsid, arguing that NS1 interacts via the structural glycoproteins with DENV particles. Results obtained with mutations residing either in the NS1 Wing domain or in the β-ladder domain suggest that NS1 might have two distinct functions in the assembly of DENV particles. By using a trans-complementation approach with a C-terminally KDEL-tagged ER-resident NS1, we demonstrate that the secretion of NS1 is dispensable for both RNA replication and infectious particle production. In conclusion, our results provide an extensive genetic map of NS1 determinants essential for viral RNA replication and identify a novel role of NS1 in virion production that is mediated via interaction with the structural proteins. These studies extend the list of NS1 functions and argue for a central role in coordinating replication and assembly/release of infectious DENV particles.