Changes in polyamine levels during aging were measured in 3-, 10- and 26-week-old female mice. The level of polyamines in pancreas, brain, and uterus was maintained during these periods. The level of spermidine slightly decreased in intestine, and decreased significantly in thymus, spleen, ovary, liver, stomach, lung, kidney, heart and muscle during these periods. In skin, the level of spermidine was maximal in 10-week-old mice and markedly reduced in 26-week-old mice. The results suggest that maintenance of polyamine levels may play important roles in the function of the pancreas, brain and uterus in 3- to 26-week-old mice. We next looked for polyamine-rich food materials as a dietary source of polyamines. Foods found to be rich in polyamines included wheat germ, rice bran, black rice, Philippine mango, green pepper, Japanese pumpkin, nuts, fermented pickles, pond smelt, turban shell viscera, whelk viscera, salted salmon roe, salted cod roe, beef intestine (boiled) and liver of eel, beef, pork and chicken; and, as previously reported, soybean, fermented soybean (natto), mushrooms, orange and green tea leaf. These results offer useful information when it becomes necessary to ingest polyamines from food.

To maintain various T cell responses and immune equilibrium, activation signals triggered by T cell antigen receptor (TCR) must be regulated by inhibitory signals. Gab2, an adaptor protein of the insulin receptor substrate-1 family, has been shown to be involved in the downstream signaling from cytokine receptors. We investigated the functional role of Gab2 in TCR-mediated signal transduction. Gab2 was phosphorylated by ZAP-70 and co-precipitated with phosphoproteins, such as ZAP-70, LAT, and CD3, upon TCR stimulation. Overexpression of Gab2 in Jurkat cells or antigen-specific T cell hybridomas resulted in the inhibition of NF-AT activation, interleukin-2 production, and tyrosine phosphorylation. The structure-function relationship of Gab2 was analyzed by mutants of Gab2. The Gab2 mutants lacking SHP-2-binding sites mostly abrogated the inhibitory activity of Gab2, but its inhibitory function was restored by fusing to active SHP-2 as a chimeric protein. A mutant with defective phosphatidylinositol 3-kinase binding capacity also impaired the inhibitory activity, and the pleckstrin homology domain-deletion mutant revealed a crucial function of the pleckstrin homology domain for localization to the plasma membrane. These results suggest that Gab2 is a substrate of ZAP-70 and functions as a switch molecule toward inhibition of TCR signal transduction by mediating the recruitment of inhibitory molecules to the TCR signaling complex. T cells recognize antigens (Ag)1 presented by the major histocompatibility complex on antigen-presenting cells (APC). An Ag recognition signal is then transmitted to the cytoplasm via the T cell antigen receptor (TCR) complex and induces T cell activation, leading to various cellular events such as proliferation, apoptosis, anergy, and differentiation as well as a variety of effector functions.To evoke appropriate T cell responses and maintain immune equilibrium, these stimulatory signals must be regulated by negative signals delivered via inhibitory molecules (1). In contrast to positive regulation, however, the precise mechanism of negative regulation of T cells has not as yet been widely characterized. In B cells and NK cells, several inhibitory receptors containing immunoreceptor tyrosine-based inhibitory motif such as Fc␥RIIB, CD22, CD72, killer inhibitory receptor, and Ly-49 have been reported (2). Upon stimulation by ligand binding, these inhibitory receptors become tyrosine-phosphorylated and recruit Src homology 2 (SH2) domain-containing tyrosine phosphatases (SHP-1 and SHP-2) and/or Src homology 2 domain-containing inositol-5-phosphatase, resulting in the inhibition of antigen receptor-mediated effector function and cytokine expression. In T cells, it has been well established that CTLA-4 and PD-1 are induced upon T cell activation and function as an inhibitory co-receptor at a late activation phase (3, 4). However, there has been little investigation of the inhibitory machineries through typical inhibitory receptors in T cells, suggesting the possibility that another inhibitory...

A functional cDNA cloning system was developed by using a retrovirus library encoding CD8-chimeric proteins and a nuclear factor of activated T cells (NFAT)-GFP reporter cell line to identify molecules inducing NFAT activation. By using this strategy, NFAT activating molecule 1 (NFAM1) was cloned as an immunoreceptor tyrosine-based activation motif (ITAM)-bearing cell surface molecule belonging to the Ig superfamily and is predominantly expressed in spleen B and T cells. NFAM1 crosslinking induced ITAM phosphorylation, ZAP-70͞Syk recruitment, NFAT activation, and cytokine production. The mechanism underlying ITAM-dependent signal transduction for lymphocyte activation is well established. Two tyrosines within ITAM are phosphorylated by Src-family kinases upon receptor engagement. Then, ZAP-70/Syk is recruited for binding to the phospho-ITAM and activated (6-8), which transduces a downstream signaling cascade by phosphorylating various adaptor͞effector molecules. Thus, ITAM represents a hallmark of signal modules that convert an Ag recognition event to initiate a signal cascade. The activation of ZAP-70͞Syk induces the activation of calcineurin and nuclear factor of activated T cells (NFAT). NFAT plays important roles in the activation of B, T, and NK cells (9-12). NFAT is rapidly dephosphorylated and translocated into the nucleus upon stimulation, and induces the transcription of various cytokine genes. Therefore, NFAT can be used as a standard readout for lymphocyte activation.We have developed a functional cDNA cloning system [NFAT-activation molecule cloning system (NACS)] for identifying molecules that induce NFAT activation by using a retrovirus cDNA library encoding CD8-chimeric molecules and reporter cells expressing NFAT-GFP, and have succeeded in identifying various cDNA clones that induce NFAT activation, including known surface molecules, kinases, adaptors, and unknown molecules. In particular, we have cloned an ITAM ϩ molecule, NFAM1 (NFAT activating molecule 1). We describe herein that NFAM1 crosslinking induces ITAM phosphorylation, Ca 2ϩ inf lux, and NFAT activation. NFAM1 co-crosslinking in B cells enhances BCR signaling, and its in vivo overexpression results in severe impairment of B cell development.The results suggest that NFAM1 belongs to a category of ITAM ϩ molecules that regulate B cell signaling and development. The successful cloning of various molecules by NACS indicates its applicability to the systematic cloning of functional molecules involved in a defined signaling pathway. Materials and MethodsMice. C57BL͞6 mice were purchased from Nihon SLC (Hamamatsu, Japan). NFAM1-transgenic (Tg) mice were generated by injecting the insert of the pHSE3Ј expression vector (provided by H. Pircher, University of Freiburg, Freiburg, Germany) (13) containing the H-2K b promoter and the IgH enhancer in which mouse NFAM1 cDNA was subcloned.NFAT-GFP Reporter Cell Line. NFAT-GFP construct was prepared by fusing three tandem NFAT-binding sites with enhanced GFP cDNA. T hybridoma 2B4 (14) was transfected ...

1 The effects of saponin from Ginseng Radix rubra on angiogenesis (tube formation) and its key steps (protease secretion, proliferation and migration) in human umbilical vein endothelial cells (HUVEC) were examined to elucidate the mechanism of the tissue repairing effects of Ginseng Radix rubra. The effect on a wound healing model was also studied. 2 Tube formation was measured by an in vitro system. The activity and immunoreactivity of tissue-type plasminogen activator (tPA) as a protease for angiogenesis and the immunoreactivity of its inhibitor, plasminogen activator inhibitor-i (PAI-1), were measured in conditioned medium of HUVEC stimulated for 24 h with saponin. Cell proliferation was measured by counting the cell numbers at 2-7 days after seeding. Migration was measured by Boyden's chamber method. The effect on wound healing was studied in the skin of diabetic rats. 3 Saponin at 10-100 gg ml-' significantly stimulated tube formation by HUVEC in a dose-dependent manner. Saponin in a similar concentration-range increased the secretion of tPA from HUVEC as estimated by immunoreactivity and enzyme activity. On the other hand, PAI-1 immunoreactivity was slightly increased at 10 gg ml-' of saponin, but then was significantly decreased at 50 and 100 jig ml-'. Cell proliferation was only slightly enhanced by 1-100 jg ml-' of saponin, but migration was significantly enhanced by 10-100 jg ml-' in a dose-dependent manner. Moreover, saponin stimulated wound healing with enhanced angiogenesis in vivo. 4 These results indicate that saponin stimulates tube formation mainly by modifying the balance of protease/protease inhibitor secretion from HUVEC and enhancing the migration of HUVEC, and that it is effective in vivo.

FcγRIII is involved in Ab-dependent cell-mediated cytotoxicity (ADCC) and cytokine production by NK cells. Signaling and expression of FcγRIII are dependent on FcRγ. Although NK cells express not only FcRγ but also CD3ζ, the role of CD3ζ in NK cell function remains unclear. Here, we found that the expression of FcγRIII on NK cells from CD3ζ-deficient mice is unexpectedly up-regulated compared with that on cells from normal mice. Furthermore, ADCC and IFN-γ production upon FcγRIII-cross-linking by NK cells from CD3ζ-deficient mice were also up-regulated. Up-regulation of the surface expression of FcγRIII on CD3ζ-deficient NK cells is not mediated by transcriptional augmentation of either FcγRIII or FcRγ gene because there was no significant difference in the expression of mRNA for FcγRIII and FcRγ. Transfection of CD3ζ into a cell line expressing FcγRIII and FcRγ induced a decrease in the cell surface expression of FcγRIII. These findings reveal a negative regulatory role of CD3ζ in FcγRIII-mediated function of murine NK cells.