Biomedical subjects

John D Jackson

Publications and source records attributed to John D Jackson.

14 recordsLinked to original sources

PubMed2006

CDD: a conserved domain database for interactive domain family analysis.

The conserved domain database (CDD) is part of NCBI's Entrez database system and serves as a primary resource for the annotation of conserved domain footprints on protein sequences in Entrez. Entrez's global query interface can be accessed at http://www.ncbi.nlm.nih.gov/Entrez and will search CDD and many other databases. Domain annotation for proteins in Entrez has been pre-computed and is readily available in the form of 'Conserved Domain' links. Novel protein sequences can be scanned against CDD using the CD-Search service; this service searches databases of CDD-derived profile models with protein sequence queries using BLAST heuristics, at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. Protein query sequences submitted to NCBI's protein BLAST search service are scanned for conserved domain signatures by default. The CDD collection contains models imported from Pfam, SMART and COG, as well as domain models curated at NCBI. NCBI curated models are organized into hierarchies of domains related by common descent. Here we report on the status of the curation effort and present a novel helper application, CDTree, which enables users of the CDD resource to examine curated hierarchies. More importantly, CDD and CDTree used in concert, serve as a powerful tool in protein classification, as they allow users to analyze protein sequences in the context of domain family hierarchies.

Amino Acid Sequence↗

PubMed2005

Tolerance by selective in vivo expansion of foreign major histocompatibility complex-transduced autologous bone marrow.

BACKGROUND: Application of gene therapy to induce antigen-specific immune tolerance could be important for transplantation or treatment of autoimmune diseases. Hematopoietic stem cell-based gene therapy has been hampered by relatively weak gene expression in vivo and loss of transduced cells over time. Selective expansion of transduced hematopoietic stem cells has been accomplished by incorporating the dihydrofolate reductase (DHFR) gene into the gene transfer vector. METHODS: To assess whether this strategy could be applied to transplantation, we constructed a retroviral vector plasmid (KA274) containing the cDNA encoding human leukocyte antigen (HLA)-A2.1 and a tyr22 mutant DHFR and generated vesicular stomatitis virus-G-pseudotyped recombinant retrovirus by transfection into 293GPG cells. Bone marrow cells from C57BL/6 mice were infected with KA274 at a multiplicity of infection of 100, and transplanted into lethally irradiated syngeneic mice. RESULTS: After transplantation with transduced bone marrow, the proportion of peripheral blood cells expressing HLA-A2 ranged from 3.2% to 38% and increased 2- to 4.9-fold after selection for DHFR-expressing cells using trimetrexate and nitrobenzylmercaptpurine riboside 5' monophosphate. HLA-A2 expression remained above pretreatment levels throughout the study. Cytotoxic spleen cells from reconstituted mice lysed third-party HLA-B7-expressing targets but were unable to lyse HLA-A2-expressing targets. All KA274 reconstituted C57BL/6 mice accepted skin grafts from HLA-A2.1 transgenic mice for more than 245 days but rejected third-party Balb/c skin grafts in 12 days. CONCLUSION: Long-term transgene expression and immunologic tolerance to retrovirus-encoded HLA-A2, equivalent to that obtained by donor bone marrow transplantation, was accomplished, and selective expansion of transduced bone marrow cells was induced using DHFR as a selectable marker.

Animals↗

PubMed2005

Competitive equality of donor cells expressing a disparate MHC antigen following stem cell-enriched bone marrow transplantation.

INTRODUCTION: Bone marrow cells expressing foreign MHC antigens survive poorly after transplantation. Stable mixed hematopoietic chimerism requires reconstitution with a relatively large number of foreign bone marrow cells and intensive depletion of host cells. In addition, when foreign MHC-transduced autologous bone marrow cells are transplanted, prolonged hematopoietic transgene expression requires extensive host conditioning. The competitive disadvantage associated with engraftment of donor cells expressing foreign MHC antigens is thought to result from a defect in engraftment secondary to donor-host incompatibility or immunologic resistance by the host. METHODS: We used a limiting-dilution competitive repopulation assay with cells from HLA-A2.1 transgenic mice to determine whether and to what extent foreign MHC antigen expression impairs engraftment in C57BL/6 hosts. Transplants were performed with Hoechst 33342 fluorescence-sorted side population (SP) cells, a subset of bone marrow enriched for stem cells. RESULTS.: Transplantation with 250 stem cell-enriched HLA-A2.1-transgenic side population cells successfully competed with nearly 5000 host C57BL/6 side population cells to produce stable long-term mixed chimerism. There was a direct relationship between the number of transplanted donor HLA-A2-expressing cells and the percentage of HLA-A2-expressing cells in the peripheral blood of reconstituted C57BL/6 mice (r2=0.1799, P=0.031). This correlation was maintained in secondary transplant recipients. CONCLUSIONS: HLA-A2-expressing hematopoietic cells do not have an engraftment defect when transplanted into C57BL/6 hosts and immunologic resistance did not limit chimerism following lethal irradiation. These results may have relevance to understanding long-term gene expression after hematopoietic stem cell based gene therapy.

Animals↗

PubMed2005

Long-term transgene expression and survival of transgene-expressing grafts following lentivirus transduction of bone marrow side population cells.

BACKGROUND: Successful transduction of hematopoietic stem cells is essential if gene therapy is to be used clinically to induce immunologic tolerance. METHODS: Hoechst 33342 staining was used to isolate a population of bone marrow cells enriched for stem cells, termed side population (SP) cells. Murine bone marrow SP cells were transduced with HLA-A2.1-expressing VSV-G-pseudotyped lentivirus or retrovirus vectors under identical conditions. RESULTS: After transduction without prestimulating cytokines, which minimizes cell cycling and helps maintain stem cell pluripotency, the HLA-A2.1 gene was found in the DNA of 56% of CFU-GM colonies derived from lentivirus-transduced SP cells, but in only 4% of colonies derived from retrovirus-transduced SP cells. Lentivirus and retrovirus transduction including cytokine prestimulation produced the same degree of integration as that following lentivirus-transduction of non-prestimulated cells. Transplantation of 5,000 lentivirus-transduced SP cells into lethally irradiated mice resulted in long-term expression of the HLA-A2.1 transgene in peripheral blood progeny of bone marrow SP cells and prolonged skin graft survival across this class I MHC barrier until the time of animal sacrifice. CONCLUSIONS: Recombinant lentivirus, but not retrovirus vectors, effectively transduced SP cells that were not prestimulated with cytokines and lentivirus-transduced SP cells successfully repopulated lethally irradiated C57BL/6 mice, animals where there is no selective advantage to repopulation with transduced cells. Transplantation of a relatively small number of transduced SP cells led to prolonged transgene mRNA expression and antigen-specific survival of grafts expressing the foreign MHC transgene.

Animals↗

PubMed2005

CDD: a Conserved Domain Database for protein classification.

The Conserved Domain Database (CDD) is the protein classification component of NCBI's Entrez query and retrieval system. CDD is linked to other Entrez databases such as Proteins, Taxonomy and PubMed, and can be accessed at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd. CD-Search, which is available at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, is a fast, interactive tool to identify conserved domains in new protein sequences. CD-Search results for protein sequences in Entrez are pre-computed to provide links between proteins and domain models, and computational annotation visible upon request. Protein-protein queries submitted to NCBI's BLAST search service at http://www.ncbi.nlm.nih.gov/BLAST are scanned for the presence of conserved domains by default. While CDD started out as essentially a mirror of publicly available domain alignment collections, such as SMART, Pfam and COG, we have continued an effort to update, and in some cases replace these models with domain hierarchies curated at the NCBI. Here, we report on the progress of the curation effort and associated improvements in the functionality of the CDD information retrieval system.

Amino Acid Sequence↗

PubMed2005

On the (mis)use of cross-price effects to gauge the effectiveness of smokeless tobacco in smoking cessation. A comment on Bask and Melkersson.

In a recent edition of this journal Mikael Bask and Maria Melkersson suggested that moist snuff, snus to the Swedes, is not an effective tool in cigarette smoking cessation. The sole basis for this conclusion is their empirical findings concerning the cross-price elasticity of demand between cigarettes and snus. Specifically, they find that, "The cross-price elasticities are negative, which indicates that snus contributes to increased smoking. Thus, even if snus taking is less harmful than cigarette smoking, it is not advisable to encourage its use in smoking cessation programs." We find this conclusion to be unwarranted. It is based on a model estimated with inappropriate data; it is based on a misspecified model with questionable theoretical underpinnings; and no evidence is presented as to the statistical significance of the cross-price effects. Furthermore, even if we ignore these technical criticisms, their conclusions are unfounded because they are based on an inappropriate conceptual experiment. We first address some technical problems with the Bask and Melkersson analysis, and then we address the fundamental flaw in their reasoning.

Humans↗

PubMed2005

Promises and pitfalls of stem cell therapy for promotion of bone healing.

UNLABELLED: There is promise in combining stem cells with allogeneic bone matrix to promote bone healing. Murine bone marrow, peripheral blood, and compact bone cells were transplanted ectopically under the kidney capsule in mice, alone or in combination with allogeneic matrix products: powder and putty to determine their bone forming potential in comparison to transplanted femoral bone fragments and long-term cultured bone marrow cells. The end point was the amount of bone formed as determined by quantitative histology. Mononuclear cells from marrow, peripheral blood, or bone alone transplanted under the kidney capsule did not form bone. Mononuclear cell populations did not combine readily with matrix products and there was in vivo migration of the transplanted combinations. Kidney subcapsular transplanted cultured bone marrow cells formed bone in proportion to the culture period, but after 9 weeks, the extent was only 20% by area of that of similarly transplanted femoral bone fragments. An inductive stimulus for bone formation seemed necessary. Osteoprogenitor cells were not detected in significant numbers in blood unless high doses of cytokines were administered. A better definition of the optimal cell populations and manipulations required for promotion of bone healing is needed along with new (transplant) models that allow for cell tracking. Much work remains to overcome current pitfalls in the use of stem cells to promote allograft integration and bone healing. LEVEL OF EVIDENCE: Therapeutic study, Level V (expert opinion). See the Guidelines for Authors for a complete description of levels of evidence.

Animals↗

PubMed2004

A comprehensive evolutionary classification of proteins encoded in complete eukaryotic genomes.

BACKGROUND: Sequencing the genomes of multiple, taxonomically diverse eukaryotes enables in-depth comparative-genomic analysis which is expected to help in reconstructing ancestral eukaryotic genomes and major events in eukaryotic evolution and in making functional predictions for currently uncharacterized conserved genes. RESULTS: We examined functional and evolutionary patterns in the recently constructed set of 5,873 clusters of predicted orthologs (eukaryotic orthologous groups or KOGs) from seven eukaryotic genomes: Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Encephalitozoon cuniculi. Conservation of KOGs through the phyletic range of eukaryotes strongly correlates with their functions and with the effect of gene knockout on the organism's viability. The approximately 40% of KOGs that are represented in six or seven species are enriched in proteins responsible for housekeeping functions, particularly translation and RNA processing. These conserved KOGs are often essential for survival and might approximate the minimal set of essential eukaryotic genes. The 131 single-member, pan-eukaryotic KOGs we identified were examined in detail. For around 20 that remained uncharacterized, functions were predicted by in-depth sequence analysis and examination of genomic context. Nearly all these proteins are subunits of known or predicted multiprotein complexes, in agreement with the balance hypothesis of evolution of gene copy number. Other KOGs show a variety of phyletic patterns, which points to major contributions of lineage-specific gene loss and the 'invention' of genes new to eukaryotic evolution. Examination of the sets of KOGs lost in individual lineages reveals co-elimination of functionally connected genes. Parsimonious scenarios of eukaryotic genome evolution and gene sets for ancestral eukaryotic forms were reconstructed. The gene set of the last common ancestor of the crown group consists of 3,413 KOGs and largely includes proteins involved in genome replication and expression, and central metabolism. Only 44% of the KOGs, mostly from the reconstructed gene set of the last common ancestor of the crown group, have detectable homologs in prokaryotes; the remainder apparently evolved via duplication with divergence and invention of new genes. CONCLUSIONS: The KOG analysis reveals a conserved core of largely essential eukaryotic genes as well as major diversification and innovation associated with evolution of eukaryotic genomes. The results provide quantitative support for major trends of eukaryotic evolution noticed previously at the qualitative level and a basis for detailed reconstruction of evolution of eukaryotic genomes and biology of ancestral forms.

Animals↗

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↗