Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain

The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans - stereoconfiguration in the beta-position are described. The water- soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis -isoprene unit in the beta-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc- P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis -isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water- soluble analog may provide an experimental approach to assay the hypothetical "flippase" proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.      

Cell division ring, a new cell division protein and vertical inheritance of a bacterial organelle in anammox planctomycetes

Anammox bacteria are members of the phylum Planctomycetes that oxidize ammonium anaerobically and produce a significant part of the atmosphere's dinitrogen gas. They contain a unique bacterial organelle, the anammoxosome, which is the locus of anammox catabolism. While studying anammox cell and anammoxosome division with transmission electron microscopy including electron tomography, we observed a cell division ring in the outermost compartment of dividing anammox cells. In most Bacteria, GTP hydrolysis drives the tubulin-analogue FtsZ to assemble into a ring-like structure at the cell division site where it functions as a scaffold for the molecular machinery that performs cell division. However, the genome of the anammox bacterium ' Candidatus Kuenenia stuttgartiensis' does not encode ftsZ . Genomic analysis of open reading frames with potential GTPase activity indicated a possible novel cell division ring gene: kustd1438, which was unrelated to ftsZ . Immunogold localization specifically localized kustd1438 to the cell division ring. Genomic analyses of other members of the phyla Planctomycetes and Chlamydiae revealed no putative functional homologues of kustd1438, suggesting that it is specific to anammox bacteria. Electron tomography also revealed that the bacterial organelle was elongated along with the rest of the cell and divided equally among daughter cells during the cell division process.     

Photocontrol of anthocyanin biosynthesis in tomato

Juvenile anthocyanin biosynthesis has been studied in dark-grown seedlings of tomato (Lycopersicon esculentum Mill.) wild types (WTs) and photomorphogenic mutants. During a subsequent 24-hr period of monochromatic irradiation at different fluence rates of red light (R) the fluence-rate response relationships for induction of anthocyanin in all the WTs are similar, yet complex, showing a response at low fluence rates (LFRR) followed by a fluence rate-dependent high irradiance response (HIR). In the hypocotyl this response is restricted to the sub-epidermal layer of cells. The high-pigment-1 (hp-1) mutant exhibits a strong amplification of both response components. Theatroviolacea (atv) mutant shows strongest amplification of the HIR component. In contrast, a transgenic line overexpressing an oat phytochrome A gene (PHYA3 ⁺) shows a most dramatic amplification of the LFRR component. The far-red light (FR)-insensitive (fri) mutant, deficient in phytochrome A (phyA), lacks the LFRR component whilst retaining a normal HIR. The temporarily R-insensitive (tri) mutant, deficient in phytochrome B1 (phyB1) retains the LFRR, but lacks the HIR. Thehp-1,fri andhp-1,tri double mutant, exhibit amplified, yet qualitatively similar responses to the monogenicfri andtri mutants. Thefri,tri double mutant lacks both response components in R, but a residual response to blue light (B) remains. Similarly, theaurea (au) mutant deficient in phytochrome chromophore biosynthesis and presumably all phytochromes, lacks both response components in the R and FR regions of the spectrum. Experiments at other wavelengths demonstrate that while there is only a small response in the FR spectral region (729 nm) in tomato, there is an appreciable HIR response in the near FR at 704 nm, which is retained in thetri mutant. This suggests that the labile phyA pool participates in the HIR at this wavelength. The intense pigmentation (Ip) mutant appears to be specifically deficient in the B1 induced anthocyanin biosynthesis. Adult plants, grown under fluorescent light/dark cycles, show a reduction of anthocyanin content of young developing leaves upon application of supplemtary or end-of-day FR. The involvement of different phytochrome species in anthocyanin biosynthesis based on micro-injection studies into theau mutant and studies using type specific phytochrome mutants is discussed.     

C and N NMR evidence for peripheral intercalation of uniformly labeled fusogenic peptides incorporated in a biomimetic membrane

Membrane fusion requires drastic and transient changes of bilayer curvature and here we have studied the interaction of three de novo designed synthetic hydrophobic peptides with a biomimetic three-lipid mixture by solid state NMR. An experimental approach is presented for screening of peptide-lipid interactions and their aggregation, and their embedding in a biomimetic membrane system using established proton-decoupled ¹³C, ¹⁵N and proton spin diffusion heteronuclear ¹H−¹³C correlation NMR methods at high magnetic field. Experiments are presented for a set of de-novo designed fusion peptides in interaction with their lipid environment. The data provide additional support for the transmembrane model for the least fusogenic peptide, L16, while the peripheral intercalation model is preferred for the fusogenic peptides LV16 and LV16G8P9. This contributes to converging evidence that peripheral intercalation is both necessary and sufficient to trigger the fusion process for a lipid mixture close to a critical point for phase separation across the bilayer.     

Online biochemical detection of glutathione-S-transferase P1-specific inhibitors in complex mixtures

A high-resolution screening (HRS) technology is described, which couples 2 parallel enzyme affinity detection (EAD) systems for substrates and inhibitors of rat cytosolic glutathione-S-transferases (cGSTs) and purified human GST P1 to gradient reversed-phase high-performance liquid chromatography (HPLC). The cGSTs and GST P1 EAD systems were optimized and validated first in flow injection analysis (FIA) mode, and optimized values were subsequently used for HPLC mode. The IC(50) values of 8 ligands thus obtained online agreed well with the IC(50) values obtained with microplate reader-based assays. For ethacrynic acid, an IC(50) value of 1.8 +/- 0.4 microM was obtained with the cGSTs EAD system in FIA mode and 0.8 +/- 0.6 microM in HPLC mode. For ethacrynic acid with the GST P1 EAD system, IC(50) values of 6.0 +/- 2.9 and 3.6 +/- 2.8 microM were obtained in FIA and HPLC modes, respectively. An HRS GST EAD system, consisting of both the cGSTs and the GST P1 EAD system in HPLC mode in parallel, was able to separate complex mixtures of compounds and to determine online their individual affinity for cGSTs and GST P1. Finally, a small library of GST inhibitors, synthesized by reaction of several electrophiles with glutathione (GSH), was successfully screened with the newly developed parallel HRS GST EAD system. It is concluded that the present online gradient HPLC-based HRS screening technology offers new perspectives for sensitive and simultaneous screening of general cGSTs and specific GST P1 inhibitors in mixtures.     

Haemocyte reactions in WSSV immersion infected Penaeus monodon

White spot syndrome virus (WSSV) has been a major cause of shrimp mortality in aquaculture worldwide in the past decades. In this study, WSSV infection (by immersion) and behaviour recruitment of haemocytes is investigated in gills and midgut, using an antiserum against the viral protein VP28 and a monoclonal antibody recognising haemocytes (WSH8) in a double immunohistochemical staining and in addition transmission electron microscopy was applied. More WSH 8(+) haemocytes were detected at 48 and 72 h post-infection in the gills of infected shrimp compared to uninfected animals. Haemocytes in the gills and midgut were not associated with VP28-immunoreactivity. In the gills many other cells showed virus replication in their nuclei, while infected nuclei in the gut cells were rare. Nevertheless, the epithelial cells in the midgut showed a clear uptake of VP28 and accumulation in supranuclear vacuoles (SNV) at 8h post-infection. However, epithelial nuclei were never VP28-immunoreactive and electron microscopy study suggests degradation of viral-like particles in the SNV. In contrast to the gills, the midgut connective tissue shows a clear increase in degranulation of haemocytes, resulting in the appearance of WSH8-immunoreactive thread-like material at 48 and 72 h post-infection. These results indicate recruitment of haemocytes upon immersion infection in the gills and degranulation of haemocytes in less infected organs, like the midgut.     

FACIL: Fast and Accurate Genetic Code Inference and Logo

MOTIVATION: The intensification of DNA sequencing will increasingly unveil uncharacterized species with potential alternative genetic codes. A total of 0.65% of the DNA sequences currently in Genbank encode their proteins with a variant genetic code, and these exceptions occur in many unrelated taxa. RESULTS: We introduce FACIL (Fast and Accurate genetic Code Inference and Logo), a fast and reliable tool to evaluate nucleic acid sequences for their genetic code that detects alternative codes even in species distantly related to known organisms. To illustrate this, we apply FACIL to a set of mitochondrial genomic contigs of Globobulimina pseudospinescens. This foraminifer does not have any sequenced close relative in the databases, yet we infer its alternative genetic code with high confidence values. Results are intuitively visualized in a Genetic Code Logo. Availability and implementation: FACIL is available as a web-based service at http://www.cmbi.ru.nl/FACIL/ and as a stand-alone program.     

Next-Generation Sequencing of a 40 Mb Linkage Interval Reveals TSPAN12 Mutations in Patients with Familial Exudative Vitreoretinopathy

Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous retinal disorder characterized by abnormal vascularisation of the peripheral retina, often accompanied by retinal detachment. To date, mutations in three genes (FZD4, LRP5, and NDP) have been shown to be causative for FEVR. In two large Dutch pedigrees segregating autosomal-dominant FEVR, genome-wide SNP analysis identified an FEVR locus of ∼40 Mb on chromosome 7. Microsatellite marker analysis suggested similar at risk haplotypes in patients of both families. To identify the causative gene, we applied next-generation sequencing in the proband of one of the families, by analyzing all exons and intron-exon boundaries of 338 genes, in addition to microRNAs, noncoding RNAs, and other highly conserved genomic regions in the 40 Mb linkage interval. After detailed bioinformatic analysis of the sequence data, prioritization of all detected sequence variants led to three candidates to be considered as the causative genetic defect in this family. One of these variants was an alanine-to-proline substitution in the transmembrane 4 superfamily member 12 protein, encoded by TSPAN12. This protein has very recently been implicated in regulating the development of retinal vasculature, together with the proteins encoded by FZD4, LRP5, and NDP. Sequence analysis of TSPAN12 revealed two mutations segregating in five of 11 FEVR families, indicating that mutations in TSPAN12 are a relatively frequent cause of FEVR. Furthermore, we demonstrate the power of targeted next-generation sequencing technology to identify disease genes in linkage intervals.     

Major histocompatibility genes in the Lake Tana African large barb species flock: evidence for complete partitioning of class II B, but not class I, genes among different species

The 16 African large barb fish species of Lake Tana inhabit different ecological niches, exploit different food webs and have different temporal and spatial spawning patterns within the lake. This unique fish species flock is thought to be the result of adaptive radiation within the past 5 million years. Previous analyses of major histocompatibility class II B exon 2 sequences in four Lake Tana African large barb species revealed that these sequences are indeed under selection. No sharing of class II B alleles was observed among the four Lake Tana African large barb species. In this study we analysed the class II B exon 2 sequences of seven additional Lake Tana African large barb species and African large barbs from the Blue Nile and its tributaries. In addition, the presence and variability of major histocompatibility complex class I UA exon 3 sequences in six Lake Tana and Blue Nile African large barb species was analysed. Phylogenetic lineages are maintained by purifying or neutral selection on non-peptide binding regions. Class II B intron 1 and exon 2 sequences were not shared among the different Lake Tana African large barb species or with the riverine barb species. In contrast, identical class I UA exon 3 sequences were found both in the lacustrine and riverine barb species. Our analyses demonstrate complete partitioning of class II B alleles among Lake Tana African large barb species. In contrast, class I alleles remain for the large part shared among species. These different modes of evolution probably reflect the unlinked nature of major histocompatibility genes in teleost fishes.Electronic Supplementary Material Supplementary material is available for this article at .An erratum to this article can be found at