Biomedical subjects

James A Harper

Publications and source records attributed to James A Harper.

5 recordsLinked to original sources

PubMed2006

Regulation of T lymphopoiesis by Notch1 and Lunatic fringe-mediated competition for intrathymic niches.

Notch1 activation regulates T lineage commitment and early T cell development. Fringe glycosyltransferases alter the sensitivity of Notch receptors to Delta-like versus Jagged Notch ligands, but their functions in T lymphopoiesis have not been defined. Here we show that developmental stage-specific expression of the glycosyltransferase lunatic fringe (Lfng) is required for coordination of the access of T cell progenitors to intrathymic niches that support Notch1-dependent phases of T cell development. Lfng-null progenitors generated few thymocytes in competitive assays, whereas Lfng overexpression converted thymocytes into 'supercompetitors' with enhanced binding of Delta-like ligands and blocked T lymphopoiesis from normal progenitors. We suggest that the ability of Lfng and Notch1 to control progenitor competition for limiting cortical niches is an important mechanism for the homeostatic regulation of thymus size.

Animals↗

PubMed2003

Fine-tuning Notch1 activation by endocytosis and glycosylation.

Recent studies have shown that disruption of Notch1 signaling in lymphocyte progenitors (LP) inhibits T cell development and promotes B cell development in the thymus. Conversely, inappropriate activation of Notch1 in LP inhibits B cell development and causes ectopic T cell development in the bone marrow. These observations imply that Notch1 activation must be spatially regulated to ensure that LP generate B cells in the bone marrow and T cells in the thymus. However, Notch ligands are expressed in both tissues. Studies in flies and worms have revealed that Notch activation is extremely sensitive to small changes in the amount of receptor or ligand expressed, and defined multiple mechanisms that limit Notch activation to discrete cells at specific times during development. Here, we describe how some of these mechanisms might regulate Notch activity in LP during the T/B lineage decision.

Animals↗

PubMed2002

Superoxide activates mitochondrial uncoupling proteins.

Uncoupling protein 1 (UCP1) diverts energy from ATP synthesis to thermogenesis in the mitochondria of brown adipose tissue by catalysing a regulated leak of protons across the inner membrane. The functions of its homologues, UCP2 and UCP3, in other tissues are debated. UCP2 and UCP3 are present at much lower abundance than UCP1, and the uncoupling with which they are associated is not significantly thermogenic. Mild uncoupling would, however, decrease the mitochondrial production of reactive oxygen species, which are important mediators of oxidative damage. Here we show that superoxide increases mitochondrial proton conductance through effects on UCP1, UCP2 and UCP3. Superoxide-induced uncoupling requires fatty acids and is inhibited by purine nucleotides. It correlates with the tissue expression of UCPs, appears in mitochondria from yeast expressing UCP1, and is absent in skeletal muscle mitochondria from UCP3 knockout mice. Our findings indicate that the interaction of superoxide with UCPs may be a mechanism for decreasing the concentrations of reactive oxygen species inside mitochondria.

Adipose Tissue, Brown↗

PubMed2002

Artifactual uncoupling by uncoupling protein 3 in yeast mitochondria at the concentrations found in mouse and rat skeletal-muscle mitochondria.

Western blots detected uncoupling protein 3 (UCP3) in skeletal-muscle mitochondria from wild-type but not UCP3 knock-out mice. Calibration with purified recombinant UCP3 showed that mouse and rat skeletal muscle contained 0.14 microg of UCP3/mg of mitochondrial protein. This very low UCP3 content is 200-700-fold less than the concentration of UCP1 in brown-adipose-tissue mitochondria from warm-adapted hamster (24-84 microg of UCP1/mg of mitochondrial protein). UCP3 was present in brown-adipose-tissue mitochondria from warm-adapted rats but was undetectable in rat heart mitochondria. We expressed human UCP3 in yeast mitochondria at levels similar to, double and 7-fold those found in rodent skeletal-muscle mitochondria. Yeast mitochondria containing UCP3 were more uncoupled than empty-vector controls, particularly at concentrations that were 7-fold physiological. However, uncoupling by UCP3 was not stimulated by the known activators palmitate and superoxide; neither were they inhibited by GDP, suggesting that the observed uncoupling was a property of non-native protein. As a control, UCP1 was expressed in yeast mitochondria at similar concentrations to that of UCP3 and at up to 50% of the physiological level of UCP1. Low levels of UCP1 gave palmitate-dependent and GDP-sensitive proton conductance but higher levels of UCP1 caused an additional GDP-insensitive uncoupling artifact. We conclude that the uncoupling of yeast mitochondria by high levels of UCP3 expression is entirely an artifact and provides no evidence for any native uncoupling activity of the protein.

Adipose Tissue, Brown↗

PubMed2001

The basal proton conductance of skeletal muscle mitochondria from transgenic mice overexpressing or lacking uncoupling protein-3.

The ability of native uncoupling protein-3 (UCP3) to uncouple mitochondrial oxidative phosphorylation is controversial. We measured the expression level of UCP3 and the proton conductance of skeletal muscle mitochondria isolated from transgenic mice overexpressing human UCP3 (UCP3-tg) and from UCP3 knockout (UCP3-KO) mice. The concentration of UCP3 in UCP3-tg mitochondria was approximately 3 microg/mg protein, approximately 20-fold higher than the wild type value. UCP3-tg mitochondria had increased nonphosphorylating respiration rates, decreased respiratory control, and approximately 4-fold increased proton conductance compared with the wild type. However, this increased uncoupling in UCP3-tg mitochondria was not caused by native function of UCP3 because it was not proportional to the increase in UCP3 concentration and was neither activated by superoxide nor inhibited by GDP. UCP3 was undetectable in mitochondria from UCP3-KO mice. Nevertheless, UCP3-KO mitochondria had unchanged respiration rates, respiratory control ratios, and proton conductance compared with the wild type under a variety of assay conditions. We conclude that uncoupling in UCP3-tg mice is an artifact of transgenic expression, and that UCP3 does not catalyze the basal proton conductance of skeletal muscle mitochondria in the absence of activators such as superoxide.

Animals↗