Aims: Vitamin C (ascorbic acid) is thought to enhance immune function, but the mechanisms involved are obscure. We utilized an in vitro model of T-cell maturation to evaluate the role of ascorbic acid in lymphocyte development. Results: Ascorbic acid was essential for the developmental progression of mouse bone marrowderived progenitor cells to functional T-lymphocytes in vitro and also played a role in vivo. Ascorbate-mediated enhancement of T-cell development was lymphoid cell-intrinsic and independent of T-cell receptor (TCR) rearrangement. Analysis of TCR rearrangements demonstrated that ascorbic acid enhanced the selection of functional TCRab after the stage of b-selection. Genes encoding the coreceptor CD8 as well as the kinase ZAP70 were upregulated by ascorbic acid. Pharmacologic inhibition of methylation marks on DNA and histones enhanced ascorbate-mediated differentiation, suggesting an epigenetic mechanism of Cd8 gene regulation via active demethylation by ascorbate-dependent Fe 2+ and 2-oxoglutarate-dependent dioxygenases. Innovation: We speculate that one aspect of gene regulation mediated by ascorbate occurs at the level of chromatin demethylation, mediated by Jumonji C ( JmjC) domain enzymes that are known to be reliant upon ascorbate as a cofactor. JmjC domain enzymes are also known to regulate transcription factor activity. These two mechanisms are likely to play key roles in the modulation of immune development and function by ascorbic acid. Conclusion: Our results provide strong experimental evidence supporting a role for ascorbic acid in T-cell maturation as well as insight into the mechanism of ascorbate-mediated enhancement of immune function.

Eisosomes are lipid domains of the yeast plasma membrane that share similarities to caveolae of higher eukaryotes. Eisosomes harbor APC-type nutrient transporters for reasons that are poorly understood. Our analyses support the model that eisosomes function as storage compartments, keeping APC transporters in a stable, inactive state. By regulating eisosomes, yeast is able to balance the number of proton-driven APC transporters with the proton-pumping activity of Pma1, thereby maintaining the plasma membrane proton gradient. Environmental or metabolic changes that disrupt the proton gradient cause the rapid restructuring of eisosomes and results in the removal of the APC transporters from the cell surface. Furthermore, we show evidence that eisosomes require the presence of APC transporters, suggesting that regulating activity of nutrient transporters is a major function of eisosomes.

Eisosomes are furrows of the yeast plasma membrane that are involved in the regulation of nutrient transporters and membrane stress pathways. Environmental changes affect plasma membrane tension and fluidity, which change both the eisosome structure and the localization of nutrient transporters and regulatory proteins to the eisosome.

Aim: To evaluate the pharmacokinetics and efficacy of a novel somatostatin receptor 2 antagonist, ZT-01, to stimulate glucagon release in rats with type 1 diabetes (T1D). Methods:The pharmacokinetics of ZT-01 and PRL-2903 were assessed following intraperitoneal or subcutaneous dosing at 10 mg/kg. We compared the efficacy of ZT-01 with PRL-2903 to prevent hypoglycaemia during an insulin bolus challenge and under hypoglycaemic clamp conditions.Results: Within 1 hour after intraperitoneal administration, ZT-01 achieved more than 10-fold higher plasma Cmax compared with PRL-2903. Twenty-four hour exposure was 4.7Â and 11.3Â higher with ZT-01 by the intraperitoneal and subcutaneous routes, respectively. The median time to reach hypoglycaemia of more than

No abstract

In cardiac tissues, the expression of multiple connexins (Cx40, Cx43, Cx45, and Cx30.2) is a requirement for proper development and function. Gap junctions formed by these connexins have distinct permeability and gating mechanisms. Since a single cell can express more than one connexin isoform, the formation of hetero-multimeric gap junction channels provides a tissue with an enormous repertoire of combinations to modulate intercellular communication. To study further the perm-selectivity and gating properties of channels containing Cx43 and Cx45, we studied two monoheteromeric combinations in which a HeLa cell co-transfected with Cx43 and Cx45 was paired with a cell expressing only one of these connexins. Macroscopic measurements of total conductance between cell pairs indicated a drastic reduction in total conductance for mono-heteromeric channels. In terms of Vj dependent gating, Cx43 homomeric connexons facing heteromeric connexons only responded weakly to voltage negativity. Cx45 homomeric connexons exhibited no change in Vj gating when facing heteromeric connexons. The distributions of unitary conductances (γj) for both mono-heteromeric channels were smaller than predicted, and both showed low permeability to the fluorescent dyes Lucifer yellow and Rhodamine123. For both mono-heteromeric channels, we observed flux asymmetry regardless of dye charge: flux was higher in the direction of the heteromeric connexon for MhetCx45 and in the direction of the homomeric Cx43 connexon for MhetCx43. Thus, our data suggest that co-expression of Cx45 and Cx43 induces the formation of heteromeric connexons with greatly reduced permeability and unitary conductance. Furthermore, it increases the asymmetry for voltage gating for opposing connexons, and it favors asymmetric flux of molecules across the junction that depends primarily on the size (not the charge) of the crossing molecules.

2623 Introduction: A complete understanding of factors that drive T cell differentiation from hematopoietic progenitors remains a fundamental goal of hematology. T cells are key regulators and effectors in defense against infections and malignancies. Aberrations in T cell regulation play causative roles in autoimmune and graft-versus-host disease. The OP9-DL1 stromal cell line enables investigation of lymphocyte development in vitro. Lymphocyte progenitors (KLS, Thy1.1 -) harvested from murine adult bone marrow and seeded onto the OP9-DL1 stromal line can be followed through stages of maturation by immunophenotyping. We previously demonstrated that addition of a stabilized form of vitamin C, phospho-ascobate (pAsc), to culture media promoted T lineage differentiation. To address the mechanism of this effect, we employed quantitative RT-PCR and spectratyping analysis to examine mRNA expression of rearranged complementarity-determining region 3 (CDR3) polymorphisms in T cell receptor (TCR) beta variable (BV) and alpha variable (AV) genes in the presence or absence of pASC. Methods: Lymphocyte progenitor cells (KLS, Thy1.1-) were sorted from adult mouse bone marrow and 1000–2000 progenitors were seeded per well in a 24 well plate coated with OP9-DL1 stromal cells. Cultures were supplemented with IL-7 (5 ng/ml), Flt3 ligand (5 ng/mL) and SCF (100 ng/mL) plus or minus pAsc (100 mcg/mL). Cells were passaged, counted and reseeded with fresh media and supplements twice a week over a 21 day period. Immunophenotype and viability were evaluated by flow cytometry. Markers for T cell development included CD44, CD25, CD3, CD4, CD8, TCR beta chain and TCR gamma-delta chains. Total RNA from cultured cells was isolated at day 21, reverse transcribed to cDNA, and analyzed by RT-PCR for differential expression of BV and AV genes using gene-specific primers for BV1, BV4, BV8.2, BV13, AV2, and AV8 with corresponding beta constant (BC) and alpha constant (AC) primers. For spectratyping, RT-PCR amplicons were generated using BV or AV gene-specific primers for BV1, BV4, BV8.2, BV13, AV1, AV2, AV5, AV8, AV10, AV13, AV16, AV18, and AV19 with corresponding BC and AC primers. These products were then re-amplified with the same gene-specific primers but with fluorochrome-labeled nested BC or AC primers. Spectratype analysis was performed on labeled amplicons by capillary electrophoresis. Results: T cell differentiation was markedly advanced by the addition of pAsc, with the majority of cells co-expressing CD4/CD8 and TCR beta/CD3. Transfection of a functionally rearranged TCR beta gene failed to rescue cells cultured without pAsc to the double positive stage; similar results were obtained with bone marrow cells derived from TCR alpha-beta transgenic donor mice. Cells cultured with pAsc demonstrated an average 5 fold increase (5.08 ± 0.40) in expression of BV genes and an average 13 fold increase (13.46 ± 2.18) of AV genes. pAsc did not induce alterations in the spectratype distributions of BV amplicons compared those generated under non-pAsc conditions, nor to distributions derived from thymic cDNA. However, spectratype distributions of AV amplicons generated under pAsc conditions more closely resembled those derived from thymic and lymph node cDNA than distributions generated from non-pAsc conditions. Conclusions: In our in vitro model, the addition of pAsc promotes robust differentiation of adult mouse bone marrow progenitors to T cells co-expressing CD4/CD8 and alpha-beta TCR. However, the mechanism by which pAsc exerts its effect remains elusive. We suspect that pAsc enhances an already pre-programmed process. The fact that transfection of a functional TCR beta gene fails to rescue differentiation, coupled with our observation that pAsc has no effect on BV spectratypes suggests that enhancement of beta selection is not involved. Rather, the AV spectratyping data suggest that pAsc exerts its effect temporally near alpha gene rearrangement, possibly via enhancement of the TCR signal transduction cascade. Further work will include PCR microarray analysis encompassing multiple signal transduction cascades involved in hematopoietic progenitor differentiation. These findings will help develop a model for future mechanistic studies and for ex vivo expansion of immune cells for therapeutic use. Disclosures: No relevant conflicts of interest to declare.

Activation of the adrenergic system in response to hypoglycemia is important for proper recovery from low glucose levels. However, it has been suggested that repeated adrenergic stimulation may also contribute to counterregulatory failure, but the underlying mechanisms are not known. The aim of this study was to establish whether repeated activation of noradrenergic receptors in the ventromedial hypothalamus (VMH) contributes to blunting of the counterregulatory response by enhancing local lactate production. The VMH of nondiabetic rats were infused with either artificial extracellular fluid, norepinephrine (NE), or salbutamol for 3 hours/day for 3 consecutive days before they underwent a hypoglycemic clamp with microdialysis to monitor changes in VMH lactate levels. Repeated exposure to NE or salbutamol suppressed both the glucagon and epinephrine responses to hypoglycemia compared to controls. Furthermore, antecedent NE and salbutamol treatments raised extracellular lactate levels in the VMH. To determine whether the elevated lactate levels were responsible for impairing the hormone response, we pharmacologically inhibited neuronal lactate transport in a subgroup of NE-treated rats during the clamp. Blocking neuronal lactate utilization improved the counterregulatory hormone responses in NE-treated animals, suggesting that repeated activation of VMH β2-adrenergic receptors increases local lactate levels which in turn, suppresses the counterregulatory hormone response to hypoglycemia.