Biomedical subjects

Heather L Wilson

Publications and source records attributed to Heather L Wilson.

8 recordsLinked to original sources

PubMed2003

Identification of differentially expressed genes representing dendritic cell precursors and their progeny.

The development of dendritic cells (DCs) from hematopoietic progenitors is not well understood. Using a spleen-derived long-term culture (LTC) system, it has been possible to continuously generate DCs from progenitors maintained in culture. The nonadherent LTC-DC population is composed of 2 major subsets. These are the small LTC-DC or DC precursors and their progeny, the large LTC-DCs that phenotypically resemble immature DCs. In this study, subtracted cDNA libraries were generated containing sequences differentially expressed in small or large LTC-DCs. Differential screening was then used on plated library clones to select genes expressed in either the small or the large cell population. Real-time polymerase chain reaction (PCR) has been used to verify the selection procedure for several genes of particular interest. Known genes isolated from subtracted libraries were related to stages in DC development and supported previous findings regarding the function of small and large LTC-DCs. Large LTC-DCs expressed a number of immunologically important genes encoding CD86, CCR1, osteopontin, and lysozyme. Small LTC-DCs resembled progenitor DCs expressing genes related to the organization of the cytoskeleton, the regulation of antigen processing, and a number of mitochondrial and ribosomal proteins. Novel transcripts were isolated from small and large LTC-DC-subtracted libraries that could encode novel proteins important in DC development. This study describes changes in gene expression related to the development of CD11c+CD11b+ major histocompatibility complex 2 low (MHC2lo) CD8alpha- DCs from precursors in a stroma-dependent culture system in the absence of exogenous cytokines.

Animals↗

PubMed2003

Dynamics of dendritic cell development from precursors maintained in stroma-dependent long-term cultures.

Two distinct subsets of dendritic cells are produced within the non-adherent cell population of the stroma-dependent long-term culture system. These are the small subset containing dendritic cell precursors and their progeny, large long-term culture-dendritic cells, which resemble immature CD11c+CD11b+MHCIIloCD8alpha- dendritic cells. The replicative and developmental potential of cells produced in long-term culture were investigated as a model for production of dendritic cells from progenitors. Cell proliferation and apoptosis were examined by labelling with bromodeoxyuridine and Annexin-V, respectively. The developmental potential of cells was analysed following transfer on to stromal monolayers or into in vitro colony and transwell assays. Results demonstrate that small long-term culture-dendritic cells are stromal cell-dependent. In the absence of stroma, they become apoptotic and die. Furthermore, direct contact with stromal cells is necessary for the differentiation and proliferation of small precursor cells. The small cell subset contains no long-term self-renewing cells, but instead appears to contain cells committed to developing into large long-term culture dendritic cells. The large long-term culture dendritic cell subset also contains dividing cells. Survival of large long-term culture-dendritic cells is dependent on soluble stroma-derived factor(s) and not direct contact with the stromal layer. All data suggest that the long-term culture system supports dendritic cell development from a self-renewing progenitor population resident within the stroma that gives rise to committed dendritic cell precursors and immature dendritic cells.

Animals↗

PubMed2003

Murine dendritic cell development: difficulties associated with subset analysis.

Dendritic cells are bone marrow-derived professional antigen presenting cells that play major roles in both the induction of primary immune responses and tolerance. It has become clear that dendritic cells are a heterogeneous group of cells that vary in cell surface marker expression and function. Multiple dendritic cell subsets have now been defined in mouse lymphoid organs and peripheral tissues. A knowledge of the function and relationship between dendritic cell subsets will be essential for understanding the regulation of immune homeostasis, immune responses and tolerance. While an increasing number of dendritic cell progenitors are being identified, the pathways that connect them remain unclear. In addition, it is unclear whether the functional divisions reflect maturation status, subset specialization or functional plasticity in response to specific pathogen and environmental signals. This review summarizes the current knowledge about the function and lineage relationship of dendritic cell subsets. It also discusses some of the difficulties associated with dendritic cell subset analysis.

Animals↗

PubMed2003

Pharmacological and biophysical properties of the human P2X5 receptor.

We constructed a full-length human P2X5 purinoceptor cDNA by incorporating a sequence corresponding to exon 10, which is missing in cDNAs cloned previously from human tissues. We studied the functional properties by patch-clamp recording and fluorescence imaging after expression in human embryonic kidney 293 cells. ATP (1-100 microM; half-maximal current at 4 microM) elicited inward currents at -60 mV; these persisted during brief (2 s) applications but declined during longer applications. The peak current was dependent on the holding potential and showed little rectification; however, both the desensitization during the application and the decline in the current when ATP was washed out were slower at +30 mV than at -60 mV. 2',3'-O-(4-Benzoyl)-benzoyl-ATP and alphabeta-methylene-ATP mimicked the action of ATP (half-maximal concentrations 6 and 161 microM, respectively). The currents were inhibited by suramin, pyridoxal-5-phosphate-6-azo-2',4'-disulfonic acid and Brilliant Blue G, with half-maximal inhibition at 3, 0.2, and 0.5 microM, respectively; 2',3'-O-(2',4',6'-trinitrophenol)-ATP (1 microM) was ineffective. Removing divalent cations did not significantly alter ATP concentration-response curves. Reversal potential measurements showed that the human P2X5 receptor was permeable to calcium (PCa/PNa = 1.5) and N-methyl-d-glucamine (NMDG) (PNMDG/PNa = 0.4); it was also permeable to chloride (PCl/PNa = 0.5) but not gluconate (Pgluc/PNa = 0.01) ions. The permeability to NMDG developed as quickly as the channel opened, in contrast to the P2X7 receptor where the NMDG permeability develops over several seconds. Cells expressing human P2X5 receptors also rapidly accumulated the propidium dye YO-PRO-1 in response to ATP.

Adenosine Triphosphate↗

PubMed2002

Role of conserved tyrosine 343 in intramolecular electron transfer in human sulfite oxidase.

Tyrosine 343 in human sulfite oxidase (SO) is conserved in all SOs sequenced to date. Intramolecular electron transfer (IET) rates between reduced heme (Fe(II)) and oxidized molybdenum (Mo(VI)) in the recombinant wild-type and Y343F human SO were measured for the first time by flash photolysis. The IET rate in wild-type human SO at pH 7.4 is about 37% of that in chicken SO with a similar decrease in k(cat). Steady-state kinetic analysis of the Y343F mutant showed an increase in K(m)(sulfite) and a decrease in k(cat) resulting in a 23-fold attenuation in the specificity constant k(cat)/K(m)(sulfite) at the optimum pH value of 8.25. This indicates that Tyr-343 is involved in the binding of the substrate and catalysis within the molybdenum active site. Furthermore, the IET rate constant in the mutant at pH 6.0 is only about one-tenth that of the wild-type enzyme, suggesting that the OH group of Tyr-343 is vital for efficient IET in SO. The pH dependences of IET rate constants in the wild-type and mutant SO are consistent with the previously proposed coupled electron-proton transfer mechanism.

Amino Acid Substitution↗

PubMed2002

Different transcription factor binding arrays modulate the cAMP responsivity of the phosphoenolpyruvate carboxykinase gene promoter.

The cAMP responsiveness of the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is mediated by a cAMP response unit, which includes three CCAAT/enhancer-binding protein (C/EBPs) sites, and a cAMP response element (CRE). Because both the CRE-binding protein and several C/EBP isoforms can to bind to the CRE with similar affinity, a variety of transcription factor bindings arrays in the cAMP response unit are possible that may affect the protein kinase A (PKA) responsivity of the promoter. To explore this issue, we have designed PEPCK promoter variants that have the native cis-elements within the cAMP response unit replaced with one or more LexA- and/or GAL4-binding sites. We also engineered the corresponding C/EBP and CRE-binding protein chimeras, which have their basic region leucine zipper domains replaced with LexA or GAL4 DNA-binding domains. Using this approach, we have reconstituted the PKA responsiveness of permissive PEPCK promoters in hepatoma cells and have characterized the PKA responsivity of the promoter under defined transcription factor occupancy patterns. Furthermore, analysis of deletion mutants of C/EBPalpha indicated that the domains that mediate its constitutive and PKA-inducible activities vary depending on which cis-element it occupies on the PEPCK promoter. These results suggest that promoter context may influence which domains within a transcription factor are employed to mediate transactivation.

Animals↗

PubMed2002

Epithelial membrane proteins induce membrane blebbing and interact with the P2X7 receptor C terminus.

The binding of extracellular ATP to the P2X(7) receptor opens an integral cation-permeable channel; it also leads to membrane blebbing and, in certain immune cells, interleukin-1beta secretion and eventual death. The latter three effects are unique to the P2X(7) receptor; also unique among P2X receptors is the long intracellular C terminus of the protein. We have shown that the C-terminal domain of the P2X(7) receptor is responsible for the cell blebbing phenotype. A screen for proteins that associate with the C-terminal domain of the P2X(7) receptor and might mediate the blebbing phenotype, identified epithelial membrane protein 2 (EMP-2). The interaction between EMP-2 and P2X(7) was confirmed biochemically by co-immunoprecipitation, co-purification, and glutathione S-transferase pull-down assays, and this interaction was entirely dependent on the C-terminal domain of P2X(7). The P2X(7) receptor also interacted with the other members of the epithelial membrane protein family (EMP-1, EMP-3, and PMP-22). All four EMPs were found to be expressed in HEK-293 cells and in THP-1 monocytes, which express P2X(7) receptors. Interestingly, the constitutive overexpression of any of the EMPs in HEK-293 cells led to cell blebbing, annexin V binding, and cell death, by a caspase-dependent pathway. These findings suggest that the P2X(7) C-terminal domain associates with EMPs, and this interaction may mediate some aspects of the downstream signaling following P2X(7) receptor activation.

Amino Acid Sequence↗

PubMed2002

CCAAT/enhancer binding proteins: do they possess intrinsic cAMP-inducible activity?

CCAAT/enhancer binding proteins (C/EBPs) are transcription factors that are enriched in tissues which play a central role in energy metabolism, such as adipose and liver. Structure/function analyses of these proteins have identified several transactivation domains, some of which can physically interact with general transcription factors present in the preinitiation complex. C/EBPs are generally considered to be constitutively-acting factors, unlike other transcription factors whose activities can be regulated by covalent modification, binding of a specific ligand, etc. However, studies of the regulatory property of the phosphoenolpyruvate carboxykinase gene promoter have uncovered a role for C/EBPs in mediating cAMP responsiveness, and identified specific domains within the proteins, which mediate this effect. Interestingly, a number of other gene promoters that are activated in response to cAMP also contain binding sites for C/EBP, and these binding sites are often located within the region of the promoter that is responsible for mediating the acute responsiveness to cAMP. The evidence presented in this review provides compelling support for the hypothesis that C/EBPs have both constitutive and cAMP-inducible activities, and should be considered as a cAMP-responsive nuclear regulator.

Animals↗