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Introduction of exogenous DNA into mammalian cells represents a powerful approach for manipulating signal transduction. The available techniques, however, are limited by low transduction efficiency and low cell viability after transduction. Here we report a highly efficient molecular delivery technique, named nanotube spearing, based on the penetration of nickel-embedded nanotubes into cell membranes by magnetic field driving. DNA plasmids containing the enhanced green fluorescent protein (EGFP) sequence were immobilized onto the nanotubes, and subsequently speared into targeted cells. We have achieved an unprecedented high transduction efficiency in Bal17 B-lymphoma, ex vivo B cells and primary neurons with high viability after transduction. This technique may provide a powerful tool for highly efficient gene transfer into a variety of cells, especially the hard-to-transfect cells.

The bioenergetic response of B lymphocytes is subject to rapid changes following antigen encounter in order to provide ATP and anabolic precursors necessary to support growth. However, the pathways involved in glucose acquisition and metabolism are unknown. We find that B lymphocytes rapidly increase glucose uptake and glycolysis following B-cell antigen receptor (BCR) crosslinking. Inhibition of glycolysis blocks BCR-mediated growth. Prior to S-phase entry, glucose metabolism shifts from primarily glycolytic to include the pentose phosphate pathway. BCR-induced glucose utilization is dependent upon phosphatidylinositol 3-kinase (PI-3K) activity as evidenced by inhibition of glucose uptake and glycolysis with LY294002 treatment of normal B cells and impaired glucose utilization in B cells deficient in the PI-3K regulatory subunit p85␣. Activation of Akt is sufficient to increase glucose utilization in B cells. We find that glucose utilization is inhibited by coengagement of the BCR and Fc␥RIIB, suggesting that limiting glucose metabolism may represent an important mechanism underlying Fc␥RIIB-mediated growth arrest. Taken together, these findings demonstrate that both growth-promoting BCR signaling and growth-inhibitory Fc␥RIIB signaling modulate glucose energy metabolism. Manipulation of these pathways may prove to be useful in the treatment of lymphoproliferative disorders, wherein clonal expansion of B lymphocytes plays a role. IntroductionIn response to antigen challenge, resting B lymphocytes exit the G 0 phase of the cell cycle and undergo a period of growth before committing to genome replication. 1,2 Growth corresponds to an accumulation of cell mass that is accompanied by increased size and is linked to increased de novo macromolecular synthesis. [3][4][5] That mammalian cell growth may be necessary for genome replication underscores its importance in adaptive immunity in that the clonal expansion of antigen-specific B lymphocytes is a prerequisite for humoral immune responses. Most investigations in B cells have focused on the role of genes whose function are important for B-cell antigen receptor (BCR)-induced protein synthesis and increased cell size. 4,5 It is recognized, however, that antigen receptor-triggered macromolecular synthesis and gene expression places enormous bioenergetic demands on lymphocytes. 5,6 Therefore, one of the fundamental aspects of B-cell responses to antigen challenge that may be critical in vivo is the provision of metabolic substrates to provide ATP and anabolic precursors for cellular growth.Early studies in lectin-stimulated thymocytes highlighted the importance of glucose uptake and catabolism in providing energy and carbon for macromolecular synthesis. 7,8 Further, proliferating thymocytes meet their ATP demand mainly by glycolytic catabolism when sufficient glucose is available. 9 It is widely viewed that glucose metabolism is regulated by homeostatic mechanisms wherein mammalian cells respond to a decreased ATP/ADP ratio by adjusting nutrient uptake and catabolism t...

Itaconic acid, or methylenesuccinic acid, is not generally classified as a mammalian metabolite. Using NMR based metabolomics and 13C-labeling, we have detected itaconic acid in both macrophage-like VM-M3 and RAW 264.7 tumor cell lines as well as stimulated and unstimulated primary murine macrophages. Macrophage activation by addition of lipopolysaccharide and IFN-γ markedly increased itaconic acid production and secretion. Crude cell extracts synthesize itaconic acid via decarboxylation of cis-aconitate, indicative of a novel mammalian cis-aconitic decarboxylase activity. Our results highlight a previously unidentified biosynthetic pathway related to TCA cycle metabolism in mammalian cells and a novel metabolite that likely plays a role in macrophage-based immune response.

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