Maltooligosaccharides from JEG-3 trophoblast-like cells exhibit immunoregulatory properties.

Problem: to better understand the immunoregulatory properties of trophoblasts, we have searched for small immunologically active carbohydrates derived from intact trophoblast-like cells.

Method Of Study: using solid phase extraction coupled with HPLC and mass spectrometry methods, we have characterized a low molecular weight carbohydrate-rich fraction associated with JEG-3 cells. We have also tested the bioactivities of selected authentic oligosaccharides found in the oligosaccharide fraction.

Ceramide kinase promotes Ca2+ signaling near IgG-opsonized targets and enhances phagolysosomal fusion in COS-1 cells.

Ceramide-1-phosphate (C1P) is a novel bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). In this study, we evaluated the mechanism by which increased C1P during phagocytosis enhances phagocytosis and phagolysosome formation in COS-1 cells expressing hCERK. Stable transfectants of COS-1 cells expressing FcgammaRIIA or both FcgammaRIIA/hCERK expression vectors were created.

2,5-Deoxyfructosazine, a D-glucosamine derivative, inhibits T-cell interleukin-2 production better than D-glucosamine.

D-Glucosamine has been widely reported to have immunosuppressive actions on neutrophils, lymphocytes, and other cells of the immune system. However, under conditions used in biological experiments (e.g.

The hexosamine biosynthesis pathway negatively regulates IL-2 production by Jurkat T cells.

To test the hypothesis that the hexosamine biosynthesis pathway (HBP) affects cytokine production, we studied IL-2 production by Jurkat cells in response to PHA. We found that the HBP activator glucosamine (GlcN), but not glucose (Glc), dose-dependently reduced IL-2 production. Importantly, GlcN blocked trafficking of a GFP-NFAT chimeric protein to the nucleus of stimulated transfectants.

IFN-gamma primes RAW264 macrophages and human monocytes for enhanced oxidant production in response to CpG DNA via metabolic signaling: roles of TLR9 and myeloperoxidase trafficking.

Macrophages and monocytes are activated by CpG DNA motifs to produce NO, which is enhanced dramatically by IFN-gamma. We hypothesize that synergistic cellular responses to IFN-gamma and CpG DNA are due to cross-talk between metabolic signaling pathways of leukocytes. Adherent RAW264.

Apparent role of dynein in glucose-6-phosphate dehydrogenase trafficking in neutrophils from pregnant women.

To better understand the mechanisms of metabolic microcompartmentalization associated with neutrophil hexose monophosphate shunt activity during pregnancy, we have studied the intracellular trafficking of glucose-6-phosphate dehydrogenase (G6PDase). Microtubule motor proteins colocalize with G6PDase. Dynein inhibitors block G6PDase accumulation at the microtubule-organizing center in pregnancy cells.

Transaldolase is part of a supramolecular complex containing glucose-6-phosphate dehydrogenase in human neutrophils that undergoes retrograde trafficking during pregnancy.

Previous studies have shown that glucose-6-phosphate dehydrogenase (G6PDase) and 6-phosphogluconate dehydrogenase form a supramolecular complex in human neutrophils that undergoes retrograde trafficking in cells from pregnant women, but anterograde trafficking in cells from nonpregnant individuals. Using fluorescence resonance energy transfer techniques, we now demonstrate that transaldolase (TALase), a key regulatory enzyme in the nonoxidative branch of the hexose monophosphate shunt, is in close physical proximity with G6PDase, but not with lactate dehydrogenase, thus suggesting the formation of a TALase-G6PDase complex. Moreover, immunofluorescence microscopy demonstrated that TALase undergoes anterograde trafficking in neutrophils from nonpregnant individuals, whereas retrograde trafficking is found during pregnancy.

Molecular proximity of complement receptor type 3 (CR3) and the glycosylphosphatidylinositol-linked protein GPI-80 on neutrophils: effects of cell adherence, exogenous saccharides, and lipid raft disrupting agents.

GPI-80, a novel glycosylphosphatidylinositol (GPI)-anchored protein on polymorphonuclear leukocytes, has been reported to cooperate with CR3 in several aspects of cell function including cell activation, adhesion and migration. The present study investigates the physical proximity of CR3 and GPI-80 on living cells using resonance energy transfer (RET) techniques, which gives positive results when the separation distance is < or = 7 nm. RET from donor-labeled anti-CR3 to acceptor-labeled anti-GPI-80 was detected on adherent neutrophils, but not observed for non-adherent cells.

Identification of channels promoting calcium spikes and waves in HT1080 tumor cells: their apparent roles in cell motility and invasion.

Intracellular Ca(2+) signals have been associated with cell polarization and locomotion. As cell motility underlies metastasis, we have sought to better characterize the Ca(2+) signaling events in HT1080 fibrosarcoma cells. We have tested the hypothesis that low voltage-activated (LVA) and nonvoltage-gated (NVG) channels of HT1080 cells participate in dynamic Ca(2+)-signaling events leading to cell migration and invasion.

Pregnancy alters glucose-6-phosphate dehydrogenase trafficking, cell metabolism, and oxidant release of maternal neutrophils.

Pregnancy is associated with changes in host susceptibility to infections and inflammatory disease. We hypothesize that metabolic enzyme trafficking affects maternal neutrophil activation. Specifically, immunofluorescence microscopy has shown that glucose-6-phosphate dehydrogenase (G-6-PDase), the rate-controlling step of the hexose monophosphate shunt (HMS), is located near the cell periphery in control neutrophils but is found near the microtubule-organizing centers in cells from pregnant women.

Hexokinase translocation during neutrophil activation, chemotaxis, and phagocytosis: disruption by cytochalasin D, dexamethasone, and indomethacin.

Neutrophils expend large amounts of energy to perform demanding cell functions. To better understand energy production and flow during cell activation, immunofluorescence microscopy was employed to determine the location of the key metabolic enzyme hexokinase during various conditions. Hexokinase is translocated from the neutrophil's cytosol to its periphery in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and other activating stimuli, but not during exposure to the formyl peptide receptor antagonist N-tert-BOC-phe-leu-phe-leu-phe (Boc-PLPLP).