Highly polymorphic repeat marker within the β-amyloid precursor protein gene

We have identified a polymorphic compound dinucleotide repeat sequence in intron 1 of the -amyloid precursor protein (APP) gene on chromosome 21. Using polymerase chain reaction (PCR) amplification of the locus, designated APPivsl, we detected 13 alleles in the CEPH family members (heterozygosity = 0.69). Lod score analysis showed complete linkage of the marker to the loci D21S210 and D21221.     

Aldehyde oxidase cross-reacting material in the Aldox n,cin, mal,and lxd mutants of Drosophila melanogaster

Rocket immunoelectrophoresis was used to estimate aldehyde oxidase cross-reacting material (AO-CRM) in larval hemolymph and adult fly extracts in mutants with reduced AO enzymatic activity. Hemolymph of larvae homozygous for Aldox ⁿ, which is a mutation of the presumed structural gene for AO, contains 30% of the wild-type CRM. The demonstration of AO-CRM in Aldox ⁿ larval hemolymph is surprising since this genotype has been reported to lack CRM. By contrast, adult Aldox ⁿ flies lack detectable CRM. The other AO-deficient mutants that were examined are cin, mal, and lxd; each has appreciable levels of CRM in both larval hemolymph and adult extracts. Detection of CRM in these mutants helps to clarify conflicting reports in the literature.This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to L.W.B.     

Proteogenomic approaches for the molecular characterization of natural microbial communities

At the present time we know little about how microbial communities function in their natural habitats. For example, how do microorganisms interact with each other and their physical and chemical surroundings and respond to environmental perturbations? We might begin to answer these questions if we could monitor the ways in which metabolic roles are partitioned amongst members as microbial communities assemble, determine how resources such as carbon, nitrogen, and energy are allocated into metabolic pathways, and understand the mechanisms by which organisms and communities respond to changes in their surroundings. Because many organisms cannot be cultivated, and given that the metabolisms of those growing in monoculture are likely to differ from those of organisms growing as part of consortia, it is vital to develop methods to study microbial communities in situ. Chemoautotrophic biofilms growing in mine tunnels hundreds of meters underground drive pyrite (FeS(2)) dissolution and acid and metal release, creating habitats that select for a small number of organism types. The geochemical and microbial simplicity of these systems, the significant biomass, and clearly defined biological-inorganic feedbacks make these ecosystem microcosms ideal for development of methods for the study of uncultivated microbial consortia. Our approach begins with the acquisition of genomic data from biofilms that are sampled over time and in different growth conditions. We have demonstrated that it is possible to assemble shotgun sequence data to reveal the gene complement of the dominant community members and to use these data to confidently identify a significant fraction of proteins from the dominant organisms by mass spectrometry (MS)-based proteomics. However, there are technical obstacles currently restricting this type of "proteogenomic" analysis. Composite genomic sequences assembled from environmental data from natural microbial communities do not capture the full range of genetic potential of the associated populations. Thus, it is necessary to develop bioinformatics approaches to generate relatively comprehensive gene inventories for each organism type. These inventories are critical for expression and functional analyses. In proteomic studies, for example, peptides that differ from those predicted from gene sequences can be measured, but they generally cannot be identified by database matching, even if the difference is only a single amino acid residue. Furthermore, many of the identified proteins have no known function. We propose that these challenges can be addressed by development of proteogenomic, biochemical, and geochemical methods that will be initially deployed in a simple, natural model ecosystem. The resulting approach should be broadly applicable and will enhance the utility and significance of genomic data from isolates and consortia for study of organisms in many habitats. Solutions draining pyrite-rich deposits are referred to as acid mine drainage (AMD). AMD is a very prevalent, international environmental problem associated with energy and metal resources. The biological-mineralogical interactions that define these systems can be harnessed for energy-efficient metal recovery and removal of sulfur from coal. The detailed understanding of microbial ecology and ecosystem dynamics resulting from the proposed work will provide a scientific foundation for dealing with the environmental challenges and technological opportunities, and yield new methods for analysis of more complex natural communities.     

Community genomics in microbial ecology and evolution

It is possible to reconstruct near-complete, and possibly complete, genomes of the dominant members of microbial communities from DNA that is extracted directly from the environment. Genome sequences from environmental samples capture the aggregate characteristics of the strain population from which they were derived. Comparison of the sequence data within and among natural populations can reveal the evolutionary processes that lead to genome diversification and speciation. Community genomic datasets can also enable subsequent gene expression and proteomic studies to determine how resources are invested and functions are distributed among community members. Ultimately, genomics can reveal how individual species and strains contribute to the net activity of the community.     

Budding of PPxY-containing rhabdoviruses is not dependent on host proteins TGS101 and VPS4A

Viral matrix proteins of several enveloped RNA viruses play important roles in virus assembly and budding and are by themselves able to bud from the cell surface in the form of lipid-enveloped, virus-like particles (VLPs). Three motifs (PT/SAP, PPxY, and YxxL) have been identified as late budding domains (L-domains) responsible for efficient budding. L-domains can functionally interact with cellular proteins involved in vacuolar sorting (VPS4A and TSG101) and endocytic pathways (Nedd4), suggesting involvement of these pathways in virus budding. Ebola virus VP40 has overlapping PTAP and PPEY motifs, which can functionally interact with TSG101 and Nedd4, respectively. As for vesicular stomatitis virus (VSV), a PPPY motif within M protein can interact with Nedd4. In addition, M protein has a PSAP sequence downstream of the PPPY motif, but the function of PSAP in budding is not clear. In this study, we compared L-domain functions between Ebola virus and VSV by constructing a chimeric M protein (M40), in which the PPPY motif of VSV M is replaced by the L domains of VP40. The budding efficiency of M40 was 10-fold higher than that of wild-type (wt) M protein. Overexpression of a dominant negative mutant of VPS4A or depletion of cellular TSG101 reduced the budding of only M40-containing VLPs but not that of wt M VLPs or live VSV. These findings suggest that the PSAP motif of M protein is not critical for budding and that there are fundamental differences between PTAP-containing viruses (Ebola virus and human immunodeficiency virus type 1) and PPPY-containing viruses (VSV and rabies virus) regarding their dependence on specific host factors for efficient budding.     

Direct microbial reduction and subsequent preservation of uranium in natural near-surface sediment

The fate of uranium in natural systems is of great environmental importance. X-ray absorption near-edge spectroscopy (XANES) revealed that U(VI) was reduced to U(IV) in shallow freshwater sediment at an open pit in an inactive uranium mine. Geochemical characterization of the sediment showed that nitrate, Fe(III), and sulfate had also been reduced in the sediment. Observations of the sediment particles and microbial cells by scanning and transmission electron microscopy, coupled with elemental analysis by energy dispersive spectroscopy, revealed that uranium was concentrated at microbial cell surfaces. U(IV) was not associated with framboidal pyrite or nanometer-scale iron sulfides, which are presumed to be of microbial origin. Uranium concentrations were not detected in association with algal cells. Phylogenetic analyses of microbial populations in the sediment by the use of 16S rRNA and dissimilatory sulfite reductase gene sequences detected organisms belonging to the families Geobacteraceae and Desulfovibrionaceae. Cultivated members of these lineages reduce U(VI) and precipitate iron sulfides. The association of uranium with cells, but not with sulfide surfaces, suggests that U(VI) is reduced by the enzymatic activities of microorganisms. Uranium was highly enriched (760 ppm) in a subsurface black layer in unsaturated sediment sampled from a pit which was exposed to seasonal fluctuations in the pond level. XANES analysis showed that the majority of uranium in this layer was U(IV), indicating that uranium is preserved in its reduced form after burial.     

Astrocytes: regulation of brain homeostasis via apolipoprotein E

Astrocytes are derived from the ventricular and subventricular zones of the neural plate, though there is controversy over their derivation from astrocyte-specific precursor cells or radial glia intermediates. Astrocytes are the most abundant cell type in the brain and contribute to brain homeostasis in several ways, including buffering of extracellular K+, regulating neurotransmitter release, forming the blood-brain barrier (BBB), releasing growth factors, and regulating the brain immune response. In addition, astrocytes have been shown to release apolipoprotein E (ApoE), which has been shown to regulate neurotransmission, growth factor release, and immune responses. Due to the diverse functions of astrocytes, they may play a role in a variety of diseases such as hepatic encephalopathy, multiple sclerosis, epilepsy, and age-related diseases including Alzheimer's disease and Parkinson's disease. This review highlights many of the diverse roles played by astrocytes in regulating brain homeostasis and discusses their potential role in a variety of disorders.