IL-4 prevents the death of naive B lymphocytes through the up-regulation of antiapoptotic proteins such as Bcl-xL. Despite studies implicating glucose utilization in growth factor-dependent survival of hemopoietic cells, the role of glucose energy metabolism in maintaining B cell viability by IL-4 is unknown. We show that IL-4 triggers glucose uptake, Glut1 expression, and glycolysis in splenic B cells; this is accompanied by increased cellular ATP. Glycolysis inhibition results in apoptosis, even in the presence of IL-4. IL-4-induced glycolysis occurs normally in B cells deficient in insulin receptor substrate-2 or the p85α subunit of PI3K and is not affected by pretreatment with PI3K or MAPK pathway inhibitors. Stat6-deficient B cells exhibit impaired IL-4-induced glycolysis. Cell-permeable, constitutively active Stat6 is effective in restoring IL-4-induced glycolysis in Stat6-deficient B cells. Therefore, besides controlling antiapoptotic proteins, IL-4 mediates B cell survival by regulating glucose energy metabolism via a Stat6-dependent pathway.

We show herein that CNT-cell complexes are formed in the presence of a magnetic field. The complexes were analyzed by flow cytometry as a quantitative method for monitoring the physical interactions between CNTs and cells. We observed an increase in side scattering signals, where the amplitude was proportional to the amount of CNTs that are associated with cells. Even after the formation of CNT-cell complexes, cell viability was not significantly decreased. The association between CNTs and cells was strong enough to be used for manipulating the complexes and thereby conducting cell separation with magnetic force. In addition, the CNT-cell complexes were also utilized to facilitate electroporation. We observed a time constant from CNT-cell complexes but not from cells alone, indicating a high level of pore formation in cell membranes. Experimentally, we achieved the expression of enhanced green fluorescence protein by using a low electroporation voltage after the formation of CNT-cell complexes. These results suggest that higher transfection efficiency, lower electroporation voltage, and miniaturized setup dimension of electroporation may be accomplished through the CNT strategy outlined herein.

Background: AMP-activated protein kinase (AMPK) is a known physiological cellular energy sensor and becomes phosphorylated at Thr-172 in response to changes in cellular ATP levels. Activated AMPK acts as either an inducer or suppressor of apoptosis depending on the severity of energy stress and the presence or absence of certain functional tumor suppressor genes.

One of the major unresolved questions in B cell biology is how the B cell Ag receptor (BCR) differentially signals to transduce anergy, apoptosis, proliferation, or differentiation during B cell maturation. We now report that extracellularly regulated kinase-mitogen-activated protein kinase (Erk-MAP kinase) can play dual roles in the regulation of the cell fate of the immature B cell lymphoma, WEHI-231, depending on the kinetics and context of Erk-MAP kinase activation. First, we show that the BCR couples to an early (≤2 h) Erk-MAP kinase signal which activates a phospholipase A2 pathway that we have previously shown to mediate collapse of mitochondrial membrane potential, resulting in depletion of cellular ATP and cathepsin B execution of apoptosis. Rescue of BCR-driven apoptosis by CD40 signaling desensitizes such early extracellularly regulated kinase (Erk) signaling and hence uncouples the BCR from the apoptotic mitochondrial phospholipase A2 pathway. A second role for Erk-MAP kinase in promoting the growth and proliferation of WEHI-231 immature B cells is evidenced by data showing that proliferating and CD40-stimulated WEHI-231 B cells exhibit a sustained cycling pattern (8–48 h) of Erk activation that correlates with cell growth and proliferation. This growth-promoting role for Erk signaling is supported by three key pieces of evidence: 1) signaling via the BCR, under conditions that induce growth arrest, completely abrogates sustained Erk activation; 2) CD40-mediated rescue from growth arrest correlates with restoration of cycling Erk activation; and 3) sustained inhibition of Erk prevents CD40-mediated rescue of BCR-driven growth arrest of WEHI-231 immature B cells. Erk-MAP kinase can therefore induce diverse biological responses in WEHI-231 cells depending on the context and kinetics of activation.

Signals derived from the BCR (B-cell antigen receptor) control survival, development and antigenic responses. One mechanism by which BCR signals may mediate these responses is by regulating cell metabolism. Indeed, the bioenergetic demands of naïve B-cells increase following BCR engagement and are characterized by a metabolic switch to aerobic glycolysis; however, the signalling pathways involved in this metabolic reprogramming are poorly defined. The PKC (protein kinase C) family plays an integral role in B-cell survival and antigenic responses. Using pharmacological inhibition and mice deficient in PKCβ, we demonstrate an essential role of PKCβ in BCR-induced glycolysis in B-cells. In contrast, mice deficient in PKCδ exhibit glycolytic rates comparable with those of wild-type B-cells following BCR cross-linking. The induction of several glycolytic genes following BCR engagement is impaired in PKCβ-deficient B-cells. Moreover, blocking glycolysis results in decreased survival of B-cells despite BCR engagement. The results establish a definitive role for PKCβ in the metabolic switch to glycolysis following BCR engagement of naïve B-cells.

Oxidative stress is a deleterious force that must be combated relentlessly by aerobic organisms and is known to underlie many human diseases including atherosclerosis, Parkinson's disease, and Alzheimer's disease. Information available about the oxidative stress response has come primarily from studies using reactive oxygen species (ROS) with ill-defined locations within the cell. Thus, existing models do not account for possible differences between stress originating within particular regions of the cell. Here, oxidative stress is studied at the subcellular level using ROS-generating compounds localizing within two different organelles: the nucleus and the mitochondrion. Differences in cytotoxicity, gene expression, and survival pathway activation are detected as a function of the subcellular origin of oxidative stress, indicating that independent mechanisms are used to cope with oxidative stress arising in different cellular compartments. These comparative studies, enabled by the development of organelle-specific oxidants, examine the cellular responses to site-specific oxidative stress with heightened precision.

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D-3-Deoxy-phosphatidylinositol derivatives have cytotoxic activity against various human cancer cell lines. These phosphatidylinositols have a potentially wide array of targets in the phosphatidylinositol-3-kinase (PI3K)/Akt signaling network. To explore the specificity of these types of molecules, we have synthesized D-3-deoxy-dioctanoylphosphatidylinositol (D-3-deoxydiC 8 PI), D-3,5-dideoxy-diC 8 PI and D-3-deoxy-dioctanoylphosphatidylinositol-5-phosphate and their enantiomers, characterized their aggregate formation by novel high resolution field cycling 31 P NMR, and examined their susceptibility to phospholipase C (PLC) and their effects on the catalytic activity of PI3K and PTEN against diC 8 PI and dioctanoylphosphatidylinositol-3-phosphate substrates, respectively, as well as their ability to induce the death of the U937 human leukemic monocyte lymphoma cells. Of these molecules, only D-3-deoxy-diC 8 PI was able to promote cell death; it did so with an IC 50 of 40 μM, well below the CMC of 0.4 mM. Under these conditions, there was little inhibition of PI3K or PTEN observed in assays of recombinant enzymes (although the complete series of deoxy-PI compounds did provide insights into ligand binding by PTEN). The D-3-deoxydiC 8 PI was a poor substrate and not an inhibitor of the PLC enzymes. The in vivo results are consistent with the current thought that the PI analogue acts on Akt1 since the transcription initiation factor eIF4e, which is a downstream signaling target of the PI3K/Akt pathway, exhibited reduced phosphorylation on Ser209. Phosphorylation of Akt1 on Ser473, but not Thr308, was reduced. Since the potent cytotoxicity for U937 cells is completely lost with the L-3-deoxy-diC 8 PI as well as with modification of the hydroxyl group at the inositol C5 (either replacing the -OH with a hydrogen or phosphorylating it) in D-3-deoxy-diC 8 PI, both chirality of the phosphoinositol moiety and the hydroxyl group at C5 are major determinants of 3-deoxy-PI binding to its target in cells.