Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

BioDeepTime: A database of biodiversity time series for modern and fossil assemblages

Motivation

We have little understanding of how communities respond to varying magnitudes and rates of environmental perturbations across temporal scales. BioDeepTime harmonizes assemblage time series of presence and abundance data to help facilitate investigations of community dynamics across timescales and the response of communities to natural and anthropogenic stressors. BioDeepTime includes time series of terrestrial and aquatic assemblages of varying spatial and temporal grain and extent from the present-day to millions of years ago.

Main Types of Variables Included

BioDeepTime currently contains 7,437,847 taxon records from 10,062 assemblage time series, each with a minimum of 10 time steps. Age constraints, sampling method, environment and taxonomic scope are provided for each time series.

Spatial Location and Grain

The database includes 8752 unique sampling locations from freshwater, marine and terrestrial ecosystems. Spatial grain represented by individual samples varies from quadrats on the order of several cm² to grid cells of ~100 km².

Time Period and Grain

BioDeepTime in aggregate currently spans the last 451 million years, with the 10,062 modern and fossil assemblage time series ranging in extent from years to millions of years. The median extent of modern time series is 18.7 years and for fossil series is 54,872 years. Temporal grain, the time encompassed by individual samples, ranges from days to tens of thousands of years.

Major Taxa and Level of Measurement

The database contains information on 28,777 unique taxa with 4,769,789 records at the species level and another 271,218 records known to the genus level, including time series of benthic and planktonic foraminifera, coccolithophores, diatoms, ostracods, plants (pollen), radiolarians and other invertebrates and vertebrates. There are to date 7012 modern and 3050 fossil time series in BioDeepTime.

Software Format

SQLite, Comma-separated values.     

Estimation of heterozygosity for single-probe multilocus DNA fingerprints

In spite of the increasing application of DNA fingerprinting to natural populations and to the genetic identification of humans, explicit methods for estimation of basic population genetic parameters from DNA fingerprinting data have not been developed. Contributing to this omission is the inability to determine, for multilocus fingerprinting probes, relatively important genetic information, such as the number of loci, the number of alleles, and the distribution of these alleles into specific loci. One of the most useful genetic parameters that could be derived from such data would be the average heterozygosity, which has traditionally been employed to measure the level of genetic variation within populations and to compare genetic variation among different loci. We derive here explicit formulas for both the estimation of average heterozygosity at multiple hypervariable loci and a maximum value for this estimate. These estimates are based upon the DNA restriction-pattern matrices that are typical for fingerprinting studies of humans and natural populations. For several empirical data sets from our laboratory, estimates of average and maximal heterozygosity are shown to be relatively close to each other. Furthermore, variances of these statistics based on simulation studies are relatively small. These observations, as well as consideration of the effect of missing alleles and alternate numbers of loci, suggest that the average heterozygosity can be accurately estimated using phenotypic DNA fingerprint patterns, because this parameter is relatively insensitive to the lack of certain genetic information.      

Probing the structure of the infectious amyloid form of the prion-forming domain of HET-s using high resolution hydrogen/deuterium exchange monitored by mass spectrometry

The HET-s prion protein of Podospora anserina represents a valuable model system to study the structural basis of prion propagation. In this system, prion infectivity can be generated in vitro from a recombinant protein. We have previously identified the region of the HET-s protein involved in amyloid formation and prion propagation. Herein, we show that a recombinant peptide corresponding to the C-terminal prion-forming domain of HET-s (residues 218-289) displays infectivity. We used high resolution hydrogen/deuterium exchange analyzed by mass spectrometry to gain insight into the structural organization of this infectious amyloid form of the HET-s-(218-289) protein. Deuterium incorporation was analyzed by ion trap mass spectrometry for 76 peptides generated by pepsin proteolysis of HET-s-(218-289). By taking into account sequence overlaps in these peptides, a resolution ranging from 4-amino acids stretches to a single residue could be achieved. This approach allowed us to define highly protected regions alternating with more accessible segments along the HET-s-(218-289) sequence. The HET-s-(218-289) fibrils are thus likely to be organized as a succession of beta-sheet segments interrupted by short turns or short loops.     

Phenotypical features of long Q-T syndrome in transgenic mice expressing human Na-K-ATPase alpha(3)-isoform in hearts

To understand why the adult human heart expresses three isoforms of the sodium pump, we generated transgenic mice (TGM) with 2.3- to 5. 5-fold overexpression of the human alpha(3)-isoform of Na-K-ATPase in the heart. Hearts from the TGM had increased maximal Na-K-ATPase activity and ouabain affinity compared with control hearts, even though the density of Na-K-ATPase pump sites (of all isoforms) was similar to that of control mice. In perfused hearts, contractility both at baseline and in the presence of ouabain tended to be greater in TGM than in controls. Surface electrocardiograms in anesthetized TGM had a steeper dependence of Q-T on sinus cycle length, and Q-T intervals measured during atrial pacing were significantly longer in TGM. Q-T dispersion during sinus rhythm also tended to be longer in TGM. Thus TGM overexpressing human alpha(3)-isoform have several of the phenotypical features of human long Q-T syndrome, despite the absence of previously described mutations in Na(+) or K(+) channels.     

Non-mendelian inheritance of the HET-s prion or HET-s prion domains determines the het-S spore killing system in Podospora anserina

Two alleles of the het-s/S locus occur naturally in the filamentous fungus Podospora anserina, het-s and het-S. The het-s encoded protein can form a prion that propagates a self-perpetuating amyloid aggregate, resulting in two phenotypes for the het-s strains. The prion-infected [Het-s] shows an antagonistic interaction to het-S whereas the prion-free [Het-s*] is neutral in interaction to het-S. The antagonism between [Het-s] and het-S is seen as heterokaryon incompatibility at the somatic level and as het-S spore killing in the sexual cycle. Two different domains of the HET-s and HET-S proteins have been identified, and a structure-function relationship has been established for interactions at the somatic level. In this study, we correlate accumulation of the HET-s and HET-S proteins (visualized using GFP) during the sexual cycle with timing of het-S spore abortion. Also, we present the structure-function relationship of the HET-s domains for interactions in the sexual cycle. We show that the constructs that ensure het-s incompatibility function in somatic mycelium are also active in het-S spore killing in the sexual cycle. In addition, paternal prion transmission and het-S spore killing has been found with the HET-s(157-289) truncated protein. The consequences of the unique transition from a coenocytic to a cellular state in the sexual phase and the timing, and localization of paternal and maternal HET-s and HET-S expression that are pertinent to prion transmission, and het-S spore killing are elaborated. These data further support our previously proposed model for het-S spore killing.     

Molecular Genetics of Heterokaryon Incompatibility in Filamentous Ascomycetes

Filamentous fungi spontaneously undergo vegetative cell fusion events within but also between individuals. These cell fusions (anastomoses) lead to cytoplasmic mixing and to the formation of vegetative heterokaryons (i.e., cells containing different nuclear types). The viability of these heterokaryons is genetically controlled by specific loci termed het loci (for heterokaryon incompatibility). Heterokaryotic cells formed between individuals of unlike het genotypes undergo a characteristic cell death reaction or else are severely inhibited in their growth. The biological significance of this phenomenon remains a puzzle. Heterokaryon incompatibility genes have been proposed to represent a vegetative self/nonself recognition system preventing heterokaryon formation between unlike individuals to limit horizontal transfer of cytoplasmic infectious elements. Molecular characterization of het genes and of genes participating in the incompatibility reaction has been achieved for two ascomycetes, Neurospora crassa and Podospora anserina. These analyses have shown that het genes are diverse in sequence and do not belong to a gene family and that at least some of them perform cellular functions in addition to their role in incompatibility. Divergence between the different allelic forms of a het gene is generally extensive, but single-amino-acid differences can be sufficient to trigger incompatibility. In some instances het gene evolution appears to be driven by positive selection, which suggests that the het genes indeed represent recognition systems. However, work on nonallelic incompatibility systems in P. anserina suggests that incompatibility might represent an accidental activation of a cellular system controlling adaptation to starvation.     

As a toxin dies a prion comes to life: A tentative natural history of the [Het-s] prion

A variety of signaling pathways, in particular with roles in cell fate and host defense, operate by a prion-like mechanism consisting in the formation of open-ended oligomeric signaling complexes termed signalosomes. This mechanism emerges as a novel paradigm in signal transduction. Among the proteins forming such signaling complexes are the Nod-like receptors (NLR), involved in innate immunity. It now appears that the [Het-s] fungal prion derives from such a cell-fate defining signaling system controlled by a fungal NLR. What was once considered as an isolated oddity turns out to be related to a conserved and widespread signaling mechanism. Herein, we recall the relation of the [Het-s] prion to the signal transduction pathway controlled by the NWD2 Nod-like receptor, leading to activation of the HET-S pore-forming cell death execution protein. We explicit an evolutionary scenario in which formation of the [Het-s] prion is the result of an exaptation process or how a loss-of-function mutation in a pore-forming cell death execution protein (HET-S) has given birth to a functional prion ([Het-s]).