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Penelope M Drake,
Jay K Nathan,
Christina M Stock,
Pamela V Chang,
Marcus O Muench,
Daisuke Nakata,
J Rachel Reader, Phung Gip,
Kevin P K Golden,
Birgit Weinhold,
Rita Gerardy-Schahn,
Frederic A Troy,
Carolyn R Bertozzi
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Penelope M Drake,
Jay K Nathan,
Christina M Stock,
Pamela V Chang,
Marcus O Muench,
Daisuke Nakata,
J Rachel Reader, Phung Gip,
Kevin P K Golden,
Birgit Weinhold,
Rita Gerardy-Schahn,
Frederic A Troy,
Carolyn R Bertozzi
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Penelope M Drake,
Jay K Nathan,
Christina M Stock,
Pamela V Chang,
Marcus O Muench,
Daisuke Nakata,
J Rachel Reader, Phung Gip,
Kevin P K Golden,
Birgit Weinhold,
Rita Gerardy-Schahn,
Frederic A Troy,
Carolyn R Bertozzi
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Penelope M Drake,
Jay K Nathan,
Christina M Stock,
Pamela V Chang,
Marcus O Muench,
Daisuke Nakata,
J Rachel Reader, Phung Gip,
Kevin P K Golden,
Birgit Weinhold,
Rita Gerardy-Schahn,
Frederic A Troy,
Carolyn R Bertozzi
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Penelope M Drake,
Jay K Nathan,
Christina M Stock,
Pamela V Chang,
Marcus O Muench,
Daisuke Nakata,
J Rachel Reader, Phung Gip,
Kevin P K Golden,
Birgit Weinhold,
Rita Gerardy-Schahn,
Frederic A Troy,
Carolyn R Bertozzi
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Penelope M Drake,
Jay K Nathan,
Christina M Stock,
Pamela V Chang,
Marcus O Muench,
Daisuke Nakata,
J Rachel Reader, Phung Gip,
Kevin P K Golden,
Birgit Weinhold,
Rita Gerardy-Schahn,
Frederic A Troy,
Carolyn R Bertozzi
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ABSTRACT: Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation.
Molecular & Cellular Proteomics 12/2011; 11(4):M111.010660. · 7.40 Impact Factor
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ABSTRACT: The sleep-deprivation-induced changes in delta power, an electroencephalographical correlate of sleep need, and brain transcriptome profiles have importantly contributed to current hypotheses on sleep function. Because sleep deprivation also induces stress, we here determined the contribution of the corticosterone component of the stress response to the electrophysiological and molecular markers of sleep need in mice.
N/A SETTINGS: Mouse sleep facility.
C57BL/6J, AKR/J, DBA/2J mice.
Sleep deprivation, adrenalectomy (ADX).
Sleep deprivation elevated corticosterone levels in 3 inbred strains, but this increase was larger in DBA/2J mice; i.e., the strain for which the rebound in delta power after sleep deprivation failed to reach significance. Elimination of the sleep-deprivation-associated corticosterone surge through ADX in DBA/2J mice did not, however, rescue the delta power rebound but did greatly reduce the number of transcripts affected by sleep deprivation. Genes no longer affected by sleep deprivation cover pathways previously implicated in sleep homeostasis, such as lipid, cholesterol (e.g., Ldlr, Hmgcs1, Dhcr7, -24, Fkbp5), energy and carbohydrate metabolism (e.g., Eno3, G6pc3, Mpdu1, Ugdh, Man1b1), protein biosynthesis (e.g., Sgk1, Alad, Fads3, Eif2c2, -3, Mat2a), and some circadian genes (Per1, -3), whereas others, such as Homer1a, remained unchanged. Moreover, several microRNAs were affected both by sleep deprivation and ADX.
Our findings indicate that corticosterone contributes to the sleep-deprivation-induced changes in brain transcriptome that have been attributed to wakefulness per se. The study identified 78 transcripts that respond to sleep loss independent of corticosterone and time of day, among which genes involved in neuroprotection prominently feature, pointing to a molecular pathway directly relevant for sleep function.
Sleep 09/2010; 33(9):1147-57. · 5.05 Impact Factor
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ABSTRACT: Although the polysialyltransferase ST8Sia IV is expressed in both primary and secondary human lymphoid organs, its product, polysialic acid (polySia), has been largely overlooked by immunologists. In contrast, polySia expression and function in the nervous system has been well characterized. In this context, polySia modulates cellular adhesion, migration, cytokine response, and contact-dependent differentiation. Provocatively, these same processes are vital components of immune development and function. We previously established that mouse multipotent hematopoietic progenitors use ST8Sia IV to express polySia on their cell surfaces. Here, we demonstrate that, relative to wild-type controls, ST8Sia IV(-/-) mice have a 30% reduction in total thymocytes and a concomitant deficiency in the earliest thymocyte precursors. T-cell progenitors originate in the bone marrow and are mobilized to the blood at regular intervals by unknown signals. We performed in vivo reconstitution experiments in which ST8Sia IV(-/-) progenitors competed with wild-type cells to repopulate depleted or deficient immune subsets. Progenitors lacking polySi exhibited a specific defect in T-cell development because of an inability to access the thymus. This phenotype probably reflects a decreased capacity of the ST8Sia IV(-/-) progenitors to escape from the bone marrow niche. Collectively, these results provide evidence that polySia is involved in hematopoietic development.
Proceedings of the National Academy of Sciences 08/2009; 106(29):11995-2000. · 9.68 Impact Factor
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Penelope M Drake,
Jay K Nathan,
Christina M Stock,
Pamela V Chang,
Marcus O Muench,
Daisuke Nakata,
J Rachel Reader, Phung Gip,
Kevin P K Golden,
Birgit Weinhold,
Rita Gerardy-Schahn,
Frederic A Troy,
Carolyn R Bertozzi
[show abstract]
[hide abstract]
ABSTRACT: Polysialic acid (polySia) is a large glycan with restricted expression, typically found attached to the protein scaffold neural cell adhesion molecule (NCAM). PolySia is best known for its proposed role in modulating neuronal development. Its presence and potential functions outside the nervous systems are essentially unexplored. Herein we show the expression of polySia on hematopoietic progenitor cells, and demonstrate a role for this glycan in immune response using both acute inflammatory and tumor models. Specifically, we found that human NK cells modulate expression of NCAM and the degree of polymerization of its polySia glycans according to activation state. This contrasts with the mouse, where polySia and NCAM expression are restricted to multipotent hematopoietic progenitors and cells developing along a myeloid lineage. Sialyltransferase 8Sia IV(-/-) mice, which lacked polySia expression in the immune compartment, demonstrated an increased contact hypersensitivity response and decreased control of tumor growth as compared with wild-type animals. This is the first demonstration of polySia expression and regulation on myeloid cells, and the results in animal models suggest a role for polySia in immune regulation.
The Journal of Immunology 12/2008; 181(10):6850-8. · 5.79 Impact Factor
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ABSTRACT: Both adult neural stem cells and embryonic stem cells have shown the capacity to differentiation into multiple cell types of the adult nervous system. They will therefore serve as valuable systems for basic investigations of cell fate choice mechanisms, as well as play important future roles in applications ranging from regenerative medicine to drug screening. However, there are significant challenges remaining, including the identification of signaling factors that specify cell fate in the stem cell niche, the analysis of intracellular targets and mechanisms of these extracellular signals, and the development of ex vivo culture systems that can exert efficient control over cell function. This review will discuss progress in the identification of signaling mechanisms and culture systems that regulate neural differentiation, neuronal differentiation, and neuronal subtype specification.
Frontiers in Bioscience 02/2008; 13:21-50. · 3.52 Impact Factor
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ABSTRACT: We hypothesized that a function of sleep is to replenish brain glycogen stores that become depleted while awake. We have previously tested this hypothesis in three inbred strains of mice by measuring brain glycogen after a 6h sleep deprivation (SD). Unexpectedly, glycogen content in the cerebral cortex did not decrease with SD in two of the strains and was even found to increase in mice of the C57BL/6J (B6) strain. Manipulations that initially induce glycogenolysis can also induce subsequent glycogen synthesis thereby elevating glycogen content beyond baseline. It is thus possible that in B6 mice, cortical glycogen content decreased early during SD and became elevated later in SD. In the present study, we therefore measured changes in brain glycogen over the course of a 6 h SD and during recovery sleep in B6 mice. We found no evidence of a decrease at any time during the SD, instead, cortical glycogen content monotonically increased with time-spent-awake and, when sleep was allowed, started to revert to control levels. Such a time-course is opposite to the one predicted by our initial hypothesis. These results demonstrate that glycogen synthesis can be achieved during prolonged wakefulness to the extent that it outweighs glycogenolysis. Maintaining this energy store seems thus not to be functionally related to sleep in this strain.
Neuroscience Letters 08/2006; 402(1-2):176-9. · 2.11 Impact Factor
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ABSTRACT: We investigated whether glucocorticoids [i.e., corticosterone (Cort) in rats] released during sleep deprivation (SD) affect regional brain glycogen stores in 34-day-old Long-Evans rats. Adrenalectomized (with Cort replacement; Adx+) and intact animals were sleep deprived for 6 h beginning at lights on and then immediately killed by microwave irradiation. Brain and liver glycogen and glucose and plasma glucose levels were measured. After SD in intact animals, glycogen levels decreased in the cerebellum and hippocampus but not in the cortex or brain stem. By contrast, glycogen levels in the cortex of Adx+ rats increased by 43% (P < 0.001) after SD, while other regions were unaffected. Also in Adx+ animals, glucose levels were decreased by an average of 28% throughout the brain after SD. Intact sleep-deprived rats had elevations of circulating Cort, blood, and liver glucose that were absent in intact control and Adx+ animals. Different responses between brain structures after SD may be due to regional variability in metabolic rate or glycogen metabolism. Our findings suggest that the elevated glucocorticoid secretion during SD causes brain glycogenolysis in response to energy demands.
AJP Regulatory Integrative and Comparative Physiology 07/2004; 286(6):R1057-62. · 3.34 Impact Factor
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ABSTRACT: Sleep has been functionally implicated in brain energy homeostasis in that it could serve to replenish brain energy stores that become depleted while awake. Sleep deprivation (SD) should therefore lower brain glycogen content. We tested this hypothesis by sleep depriving mice of three inbred strains, i.e., AKR/J (AK), DBA/2J (D2), and C57BL/6J (B6), that differ greatly in their sleep regulation. After a 6-h SD, these mice and their controls were killed by microwave irradiation, and glycogen and glucose were quantified in the cerebral cortex, brain stem, and cerebellum. After SD, both measures significantly increased by approximately 40% in the cortex of B6 mice, while glycogen significantly decreased by 20-38% in brain stem and cerebellum of AK and D2 mice. In contrast, after SD, glucose content increased in all three structures in AK mice and did not change in D2 mice. The increase in glycogen after SD in B6 mice persisted under conditions of food deprivation that, by itself, lowered cortical glycogen. Furthermore, the strains that differ most in their compensatory response to sleep loss, i.e., AK and D2, did not differ in their glycogen response. Thus glycogen content per se is an unlikely end point of sleep's functional role in brain energy homeostasis.
AJP Regulatory Integrative and Comparative Physiology 09/2003; 285(2):R413-9. · 3.34 Impact Factor
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ABSTRACT: We tested whether brain glycogen reserves were depleted by sleep deprivation (SD) in Long-Evans rats 20-59 days old. Animals were sleep deprived beginning at lights on and then immediately killed by microwave irradiation. Glycogen and glucose levels were measured by a fluorescence enzymatic assay. In all age groups, SD reduced cerebellar glycogen levels by an average of 26% after 6 h of SD. No changes were observed in the cortex after 6 h of SD, but in the oldest animals, 12 h of SD increased cortical glycogen levels. There was a developmental increase in basal glycogen levels in both the cortex and cerebellum that peaked at 34 days and declined thereafter. Robust differences in cortical and cerebellar glycogen levels in response to enforced waking may reflect regional differences in energy utilization and regulation during wakefulness. These results show that brain glycogen reserves are sensitive to SD.
AJP Regulatory Integrative and Comparative Physiology 08/2002; 283(1):R54-9. · 3.34 Impact Factor
