Biomedical subjects

John D Jackson

Publications and source records attributed to John D Jackson.

6 recordsLinked to original sources

PubMed2003

The COG database: an updated version includes eukaryotes.

BACKGROUND: The availability of multiple, essentially complete genome sequences of prokaryotes and eukaryotes spurred both the demand and the opportunity for the construction of an evolutionary classification of genes from these genomes. Such a classification system based on orthologous relationships between genes appears to be a natural framework for comparative genomics and should facilitate both functional annotation of genomes and large-scale evolutionary studies. RESULTS: We describe here a major update of the previously developed system for delineation of Clusters of Orthologous Groups of proteins (COGs) from the sequenced genomes of prokaryotes and unicellular eukaryotes and the construction of clusters of predicted orthologs for 7 eukaryotic genomes, which we named KOGs after eukaryotic orthologous groups. The COG collection currently consists of 138,458 proteins, which form 4873 COGs and comprise 75% of the 185,505 (predicted) proteins encoded in 66 genomes of unicellular organisms. The eukaryotic orthologous groups (KOGs) include proteins from 7 eukaryotic genomes: three animals (the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster and Homo sapiens), one plant, Arabidopsis thaliana, two fungi (Saccharomyces cerevisiae and Schizosaccharomyces pombe), and the intracellular microsporidian parasite Encephalitozoon cuniculi. The current KOG set consists of 4852 clusters of orthologs, which include 59,838 proteins, or approximately 54% of the analyzed eukaryotic 110,655 gene products. Compared to the coverage of the prokaryotic genomes with COGs, a considerably smaller fraction of eukaryotic genes could be included into the KOGs; addition of new eukaryotic genomes is expected to result in substantial increase in the coverage of eukaryotic genomes with KOGs. Examination of the phyletic patterns of KOGs reveals a conserved core represented in all analyzed species and consisting of approximately 20% of the KOG set. This conserved portion of the KOG set is much greater than the ubiquitous portion of the COG set (approximately 1% of the COGs). In part, this difference is probably due to the small number of included eukaryotic genomes, but it could also reflect the relative compactness of eukaryotes as a clade and the greater evolutionary stability of eukaryotic genomes. CONCLUSION: The updated collection of orthologous protein sets for prokaryotes and eukaryotes is expected to be a useful platform for functional annotation of newly sequenced genomes, including those of complex eukaryotes, and genome-wide evolutionary studies.

Animals↗

PubMed2003

MMDB: Entrez's 3D-structure database.

Three-dimensional structures are now known within most protein families and it is likely, when searching a sequence database, that one will identify a homolog of known structure. The goal of Entrez's 3D-structure database is to make structure information and the functional annotation it can provide easily accessible to molecular biologists. To this end, Entrez's search engine provides several powerful features: (i) links between databases, for example between a protein's sequence and structure; (ii) pre-computed sequence and structure neighbors; and (iii) structure and sequence/structure alignment visualization. Here, we focus on a new feature of Entrez's Molecular Modeling Database (MMDB): Graphical summaries of the biological annotation available for each 3D structure, based on the results of automated comparative analysis. MMDB is available at: http://www.ncbi.nlm.nih.gov/Entrez/structure.html.

Animals↗

PubMed2003

CDD: a curated Entrez database of conserved domain alignments.

The Conserved Domain Database (CDD) is now indexed as a separate database within the Entrez system and linked to other Entrez databases such as MEDLINE(R). This allows users to search for domain types by name, for example, or to view the domain architecture of any protein in Entrez's sequence database. CDD can be accessed on the WorldWideWeb at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd. Users may also employ the CD-Search service to identify conserved domains in new sequences, at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. CD-Search results, and pre-computed links from Entrez's protein database, are calculated using the RPS-BLAST algorithm and Position Specific Score Matrices (PSSMs) derived from CDD alignments. CD-Searches are also run by default for protein-protein queries submitted to BLAST(R) at http://www.ncbi.nlm.nih.gov/BLAST. CDD mirrors the publicly available domain alignment collections SMART and PFAM, and now also contains alignment models curated at NCBI. Structure information is used to identify the core substructure likely to be present in all family members, and to produce sequence alignments consistent with structure conservation. This alignment model allows NCBI curators to annotate 'columns' corresponding to functional sites conserved among family members.

Amino Acid Sequence↗

PubMed2003

Direct identification and enrichment of retinal stem cells/progenitors by Hoechst dye efflux assay.

PURPOSE: The present study describes a method for isolating neural stem cells/progenitors directly from the freshly dissociated embryonic retina (prospective identification) and compares their characteristics with those enriched from mitogen-exposed embryonic retinal cell culture. METHODS: Cell dissociates from embryonic rat retina and mitogen-exposed embryonic retinal cultures were stained with Hoechst 33342 fluorescent dye. The emission patterns of cells were analyzed in both blue and red wavelength using flow cytometry to enrich cells that retained or excluded the dye. The phenotype characteristics and differentiation potential of enriched cells were analyzed by immunocytochemical, RT-PCR, and electrophysiological analyses. RESULTS: The Hoechst dye efflux assay identified a minor population of cells, called side population (SP) cells, in fresh retinal dissociates. These cells that preferentially excluded the Hoechst 33342 fluorescent dye were proliferative and expressed both neural progenitor and retinal progenitor markers. The retinal SP cells generated functional neurons and glia and possessed the ability to differentiate along lineages of different late-born retinal cell types. Cells of similar phenotypes and potential were observed in the SP obtained from mitogen-exposed retinal culture. CONCLUSIONS: The Hoechst dye efflux assay represents an effective method for direct identification of retinal stem cells/progenitors. These results demonstrate that the prospectively isolated retinal stem cells/progenitors and those enriched as SP cells from mitogen-exposed retinal cell culture may be similar in their properties and potential.

Animals↗

PubMed2002

Porcine hematopoiesis on primate stroma in long-term cultures: enhanced growth with neutralizing tumor necrosis factor-alpha and tumor growth factor-beta antibodies.

BACKGROUND: Donor hematopoiesis is at a competitive disadvantage when bone marrow transplantation is across species barriers. This could present major limitations to xenogeneic stem cell transplantation as an approach to tolerance induction. An in vitro model of xenogeneic engraftment was established to identify inhibitors of porcine hematopoiesis in a primate environment. METHODS: Porcine bone marrow cells (BMC), in the presence or absence of primate CD34+ positive cells, were cultured for 4-6 weeks on primate stroma with porcine cytokines. Cellularity and growth of colony-forming cells were indicators of hematopoietic growth. Effects of soluble factors were determined by using Transwell inserts to separate porcine cells from stroma. Neutralizing antibodies for human transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) were added to cultures. RESULTS: Porcine hematopoiesis can be maintained in long-term cultures on primate stroma with pig cytokines. Adding BMC to the stroma below Transwell-containing porcine cells dramatically inhibited porcine hematopoiesis, showing an inhibitory role for soluble factors. Neutralizing antibodies against TNF-alpha or TGF-beta caused a modest enhancement of porcine hematopoiesis; however, the combination of both led to a substantial increase. Inhibitory effects of these cytokines were confirmed by adding TNF-alpha and TGF-beta to porcine cultures. CONCLUSIONS: Porcine cells may be more sensitive to inhibitory effects of TNF-alpha and TGF-beta than primate cells and are at a disadvantage when in a primate environment. Potential implications of this observation are discussed in the context of establishing specific immune tolerance via mixed chimerism to facilitate xenotransplantation.

Animals↗

PubMed2002

Induction of chimerism in mice using human MHC class I-mismatched Hoechst 33342 side population donor stem cells.

A population of Hoechst 33342-stained cells, termed side population (SP) cells, can reconstitute the hematopoietic system of syngeneic mice. This study examined whether limiting numbers of SP cells can repopulate mice across a xenogeneic MHC class I barrier. SP cells were isolated from HLA.B7 and HLA.A2.1 transgenic mice by FACS and placed in colony assays or transplanted into irradiated C57BL/6 (B/6) recipients. SP cells contained few colony-forming cells when placed directly in culture. The number of GM-CFC and HPP-CFC increased up to 3000- and 300-fold, respectively, after 7 days in IL-3- and SCF-stimulated liquid culture. BMC-derived GM-CFC increased up to only 12-fold and HPP-CFC decreased after 7 days in culture. HLA-B7 SP cells (2500-5000) were transplanted into lethal-irradiated B/6 mice. Two-color flow analysis, 4-6 weeks after transplantation, showed that HLA-B7 expression in granulocyte-, macrophage-, and lymphocyte-specific lineages from reconstituted mice was similar to that in B7 transgenic mice. Secondary transplanted B/6 mice also showed a pattern of HLA-B7 expression similar to that in transgenic mice and were followed for longer than 16 weeks with stable chimerism. When HLA-A2.1 SP cells were transplanted into sublethally irradiated mice, 50% of the mice expressed HLA-A2 by PCR analysis in short-term repopulation studies. These data confirm that limiting numbers of SP cells can repopulate the major hematopoietic lineages in lethal and sublethally irradiated mice across a human MHC class I barrier. Therefore, SP cells may be useful for establishing mixed chimerism, which may induce immunologic nonresponsiveness to donor antigens in solid organ transplantation.

Animals↗