Nucleotide sequences of VJ (variable-joining) junctional regions of V14' a-chain T-cell receptor genes show that most VaW4+ T cells use one a chain (Val4Ja281 with a one-nucleotide N region, which is frequently used in keyhole limpet hemocyanin-specific suppressor T-celi hybridomas) in unprimed mice. Moreover, the frequency of this a-chain expression was >1.5% of the total a chains found in laboratory strains, including B10 congenic mice. This is about 104 times higher than was expected. The V14J281 a-chain expression was relatively low but was significant in CD41/CD81 immature thymocytes and became quite high in mature single-positive T cells, implying that this a chain is selected during T-cell maturation. V14J281 expression increased with time after birth and reached a maximum at around 5 weeks of age. The ligand seems to be a self molecule and to be present in laboratory strains but to be absent in a wild mouse, Mus musculus molossinus, because bone marrow chimeras clearly showed that bone marrow cells derived from Mus musculus molossinus negative for this a chain raised V14J281-positive T cells in a C57BL/6 environment. The above results suggest that there are some selection mechanisms for this cell type other than those for conventional afi T cells and also that the homogenous VJ junction of the V14J281 a chain plays a pivotal role in the selection of the T cell and its ligand reactivity.Thymus-derived lymphocytes (T cells) recognize antigens in the context of the polymorphic parts of major histocompatibility complex (MHC) class I or class II molecules by virtue ofthe heterodimeric a/3 T-cell receptor (TCR), which is found on the vast majority of mature T cells in the thymus and peripheral lymphoid tissues (1, 2). A second TCR, y8 TCR, has also been identified (3, 4) and found to be relatively abundant in some adult mouse organs and among fetal thymocytes.The T-cell repertoire seems to be generated by two selection mechanisms during T-cell We provide evidence herein that some T cells use a homogenous a chain (V14J281). This seems to be a consequence of selection and expansion ofthis T-cell type, because its frequency is >1.5% of the total a chains in the peripheral lymphoid organs. Moreover, studies using bone marrow chimeras suggest that the ligand is an unidentified self molecule. The results indicate that the homogenous VJjunctional region of the V14J281 a chain, in particular, plays a crucial role in the selection of this type of T cell and its ligand reactivity. MATERIALS AND METHODSAnimals and Cell Lines. Pathogen-free C57BL/6 mice were purchased from Shizuoka Experimental Animal Co., Hamamatsu, Japan. A wild Japanese mouse strain, Mus musculus molossinus, was established and maintained by K.M. Other strains, including B10 congenic lines used, were also maintained by K.M. A thymoma cell line of AKR origin, BW5147, and a keyhole limpet hemocyanin (KLH)-specific suppressor T-cell (Ts) hybridoma (BW5147 x C57BL/6 Ts; 34S-281) used in the present studies have been described (12,13

The CD15 carbohydrate epitope is expressed in mature human neutrophils, monocytes, and promyelocytes. We aimed to determine the ␣1,3-fucosyltransferase responsible for the expression of CD15 in each subpopulation of leukocytes. Three ␣1,3-fucosyltransferases, FUT4, FUT7, and FUT9, are expressed in human leukocytes. We demonstrated that FUT9 exhibits 20-fold stronger activity for CD15 synthesis than FUT4, whereas FUT4 exhibits 4.5-fold stronger activity for CDw65 synthesis than FUT9. By competitive reverse transcriptase-polymerase chain reaction, FUT9 was found to be strongly expressed in mature granulocytes and peripheral blood mononuclear cell, but not in monocytes. CD34؉ and CD15 ؉ cells in cord blood and myeloid cell lines (HL-60 and U937) did not express FUT9 at all. FUT4 transcripts were ubiquitously expressed in all blood cells and all cultured cell lines, with HL-60 and U937 cells in particular expressing a number of FUT4 transcripts. Transfection of the FUT9 gene into Jurkat and U937 cells demonstrated that FUT9 has the potential to express CD15 in myeloid and lymphoid cells. These findings suggest that the expression of CD15 in mature granulocytes is directed by FUT9, whereas it is determined in promyelocytes and monocytes by FUT4. Measurement of CD15 synthesizing activity in cell homogenates of each cell population using the polylactosamine acceptor further supported these conclusions.There are three CD 1 markers of human leukocytes comprising fucosylated carbohydrate epitopes. As listed in Fig. 1 below, the distal lactosamine unit (LN; type 2 chain), Gal␤1,4GlcNAc, of the polylactosamine chain is fucosylated through ␣1,3-fucosyltransferase (␣1,3FUT) activity to form the CD15 (Lewis x; Le X ) epitope (1, 2). The CD15s (sialylated CD15; sialyl Le X (sLe X )) and CDw65 (VIM-2) epitopes are also fucosylated structures related to CD15, i.e. CD15s is formed by ␣2,3-sialylation prior to the fucosylation of the distal LN unit of polylactosamine by ␣1,3FUT, and CDw65 is formed by fucosylation of the inner LN unit of ␣2,3-sialylated polylactosamine by ␣1,3FUT (2, 3).The CD15 epitope is expressed in some tissues, such as epithelial cells of intestinal tissues (4 -6), certain neurons and glial cells in the central nervous system (7,8). In human leukocytes, CD15 is expressed preferentially in monocytes, mature neutrophils, and all myeloid cells from the promyelocyte stage onwards, making it a useful cell surface marker (9 -11). CD15 is considered to be involved in neutrophil functions, that is, cell-cell interactions, phagocytosis, stimulation of degranulation, and respiratory burst, although the function of CD15 is not clear (12)(13)(14)(15)(16).Six human ␣1,3FUT genes have been cloned to date, which are FUT3 (Fuc-TIII), FUT4 (Fuc-TIV), FUT5 (Fuc-TV), FUT6 (Fuc-TVI), FUT7 (Fuc-TVII), and FUT9 (Fuc-TIX) (1, 17-23). FUT9, a new member of the human ␣1,3FUT family, which we have recently cloned, is expressed in human leukocytes, glandular compartments of the stomach, and forebrain (23). The FUT9 gene was mapped on ch...

Human promonocytic cell line U937 cells can be induced to differentiate into macrophages by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA treatment induced the expression of the monocytic differentiation markers CD11b and CD36, with concomitant morphological changes. Moreover, TPA enhanced reactive oxygen species (ROS) generation in these cells, and phagocytic ability was also stimulated during differentiation. The antioxidant agent N-acetyl-L-cysteine inhibited the TPA-induced differentiation of U937 cells. TPA treatment decreased the expression level of catalase, which catalyzes the decomposition of hydrogen peroxide (H 2 O 2 ) to H 2 O and O 2 . In contrast, TPA increased the level of manganese superoxide dismutase, which catalyzes the dismutation of superoxide into H 2 O 2 and O 2 without affecting the levels of copper-zinc superoxide dismutase or glutathione peroxidase 1, which removes H 2 O 2 using glutathione as substrate. Treatment of U937 cells with catalase inhibited the enhancement of ROS generation induced by TPA, and blocked the TPA-induced differentiation of U937 cells. Human promyelocytic cell line HL60 cells were also induced to differentiate into macrophages by TPA. However, HP100-1 cells, its variant cell line overexpressing catalase, were resistant to TPA-induced differentiation. Our results suggest that catalase inhibits monocytic differentiation by TPA; the decrease in catalase level and the accumulation of H 2 O 2 are significant events for monocyte/ macrophage differentiation by TPA.

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Cancer cells often develop drug resistance. In cisplatin-resistant HeLa cisR cells, fibroblast growth factor 13 (FGF13/FHF2) gene and protein expression was strongly upregulated, and intracellular platinum concentrations were kept low. When the FGF13 expression was suppressed, both the cells' resistance to platinum drugs and their ability to keep intracellular platinum low were abolished. Overexpression of FGF13 in parent cells led to greater resistance to cisplatin and reductions in the intracellular platinum concentration. These cisplatin-resistant cells also showed increased resistance to copper. In preoperative cervical cancer biopsy samples from poor prognoses patients after cisplatin chemoradiotherapy, FGF13-positive cells were detected more abundantly than in the biopsy samples from patients with good prognoses. These results suggest that FGF13 plays a pivotal role in mediating resistance to platinum drugs, possibly via a mechanism shared by platinum and copper. Our results point to FGF13 as a novel target and useful prognostic guide for cancer therapy.

Cutaneous lymphocyte-associated antigen (CLA), which plays a key part in skin homing of human CD4+ memory T cells via CLA/E-selectin binding, is upregulated by IL-12 and downregulated by IL-4. Although alpha1,3-fucosyltransferase VII is essential for synthesis of the CLA carbohydrate epitope, little is known about how the CLA expression is regulated by a number of glycosyltransferases. A 6 wk long-term culture for the in vitro differentiation of naïve Th cells to memory Th1 cells was employed. By repeated activation in the presence of IL-12, naïve T cells differentiated into memory Th1 cells, resulting in the upregulation of CLA expression. The switching of cytokine from IL-12 to IL-4 at three cycles resulted in a marked downregulation of CLA. The transcript levels of 16 glycosyltransferases and P-selectin glycoprotein ligand-1, all considered to be potentially involved in CLA synthesis, were determined after each cycle. The level of CLA expression was well correlated with the amounts of alpha1,3-fucosyltransferase VII and beta1,4-galactosyltransferase I. Both were upregulated by IL-12 and downregulated by IL-4. In particular, alpha1,3-fucosyltransferase VII levels decreased markedly in the presence of IL-4. P-selectin glycoprotein ligand-1 and Core 2 beta1, 6-N-acetylglucosaminyltransferase were progressively up-regulated by repeated IL-12 stimulation, but they were not downregulated by IL-4. The transcript levels of some genes examined were constitutive without any correlation to CLA expression. These results suggest that the level of CLA expression is determined by alpha1, 3-fucosyltransferase VII and beta1,4-galactosyltransferase I, the other enzymes merely participating in the synthesis of CLA. In peripheral blood mononuclear cells, IL-12 and IL-4 profoundly upregulated and downregulated the alpha1,3-fucosyltransferase VII transcripts, respectively, but not the beta1,4-galactosyltransferase I ones, within only 2 h of in vitro culture. This suggested that alpha1,3-fucosyltransferase VII is transcriptionally regulated directly by IL-12 and IL-4.

The extracellular effect of fibroblast growth factor-12 (FGF12) remains unknown because FGF12 cannot activate any fibroblast growth factor receptors (FGFRs), and FGF12 is not currently thought to be released from cells. We reported previously that FGF12 plays an intracellular role in the inhibition of radiation-induced apoptosis. In this study, we demonstrated that recombinant FGF12 was able to be internalized into the cytoplasm of a rat intestinal epithelial cell line, IEC6, and this process was dependent on two novel cell-penetrating peptide (CPP) domains (CPP-M and CPP-C). In particular, CPP-C, composed of ϳ10 amino acids, was identified as a specific domain of FGF12 and its subfamily in the C-terminal region (residues 140 -149), although CPP-M was a common domain in the internal region of the FGF family. The absence of CPP-C from FGF12 or a mutation (E142L) in the CPP-C domain drastically reduced the internalization of FGF12 into cells. Therefore, CPP-C played an essential role in the internalization of FGF12. In addition, CPP-C was able to deliver other polypeptides into cells as a CPP because an FGF1/CPP-C chimeric protein was internalized into IEC6 cells more efficiently than wild-type FGF1. Finally, intraperitoneally added FGF12 inhibited radiation-induced apoptosis in the intestinal epithelial cells of BALB/c mice, and deletion of the CPP-C domain decreased the inhibition of the apoptosis. These findings suggest that exogenous FGF12 can play a role in tissues by translocating into cells through the plasma membrane, and the availability of this novel CPP provides a new tool for the intracellular delivery of bioactive molecules.

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.