The termination of protein synthesis in bacteria requires two codon-specific polypeptide release factors, RF-1 and RF-2. A third factor, RF-3, which stimulates the RF-1 and RF-2 activities, was originally identified in Escherichia coli, but it has received little attention since the 1970s. To search for the gene encoding RF-3, we selected nonsensesuppressor mutations by random insertion mutagenesis on the assumption that a loss of function of RF-3 would lead to misreading of stop signals. One ofthese mutations, named tos-l (for transposon-induced opal suppressor), mapped to the 99.2 min region on the E. coli chromosome and suppressed all three stop codons. Complementation studies and analyses ofthe DNA and protein sequences revealed that the tos gene encodes a 59,442-Da protein, with sequence homology to elongation factor EF-G, including G-domain motifs, and that the tos-l insertion eliminated the C-terminal one-fifth of the protein.Extracts containing the overproduced Tos protein markedly increased the formation of ribosomal termination complexes and stimulated the RF-1 or RF-2 activity in the codondependent in vitro termination assay. The stimulation was fcantly reduced by GTP, GDP, and the 13,-methylene analog ofGTP, but not by GMP. These results fit perfectly with those described in the original publications on RF-3, and the tos gene has therefore been designatedpC. A completely nullprJC mutation made by reverse genetics affected the cell growth under the limited set of physiological and strain conditions. The termination oftranslation in bacteria requires two codonspecific peptide-chain-release factors: release factor 1 (RF-1; UAG/UAA-specific) and release factor 2 (RF-2; UGA/ UAA-specific) (1). Stop codon recognition by release factors holds considerable interest, since it entails protein-RNA recognition rather than a codon-anticodon (RNA-RNA) interaction, but the mechanism is still unknown (2, 3).The genes encoding the Escherichia coli release factors RF-1 and RF-2 have been isolated (4, 5). The map position of RF-1 (designated prfA) is 27 min on the E. coli chromosome (6), and RF-2 (designated prfB) is at 62 min, within the same operon as the lysyl-tRNA synthetase gene (7). Several mutants of RF-1 and RF-2 have been isolated, and they often cause misreading of stop codons or frameshifting, as well as temperature-sensitive growth of the cells (8-11). Hence the reduced activity of release factors results in several translational errors in vivo, and these errors are probably caused by an abnormally long pausing of ribosomes at stop signals (12).In E. coli, a third factor, RF-3, is known to stimulate the activities of RF-1 and RF-2; it binds guanine nucleotides but is not codon-specific (13,14). This factor has received little attention since its initial characterization. RF-3 was shown to partially correct the very poor binding of RE-1 and RF-2 to L7/L12-depleted ribosomes (15). To investigate the biological significance and function of RF-3 in stop codon recognition, polypeptide release, and cell g...

Human V␣24 NKT cells bearing an invariant V␣24J␣Q antigen receptor, the counterpart of murine V␣14 NKT cells, are activated by a specific ligand, ␣-GalCer, in a CD1d-dependent manner. Here, we demonstrate decreased numbers of circulating V␣24 NKT cells in patients with primary lung cancer compared to healthy volunteers. However, V␣24 NKT cells and DCs from lung cancer patients were functionally normal, even in the presence of tumor. Furthermore, levels of V␣24 NKT cells in surgically resected lung tissue appeared to be equivalent to those of V␣14 NKT cells in the mouse lung. Levels of V␣24 NKT cells in the tumor tissue itself were increased about 2.5 times. Administration of ␣-GalCer-pulsed DCs expanded V␣14 NKT cells in the lung more than 10 times, and the increased levels were sustained for 1 week. This may explain the previous finding that ␣-GalCer-pulsed DCs exerted strong antitumor activity in mouse lung tumor metastatic models. The potential use of ␣-GalCer-pulsed DCs for immunotherapy aimed at activating endogenous V␣24 NKT cells in the lung of cancer patients is discussed. © 2002 Wiley-Liss, Inc. Key words: V␣24 NKT cell; IFN-␥ primary lung cancer; ␣-galactosylceramide; single-cell sorting RT-PCRMurine V␣14 NKT cells are characterized by coexpression of NK1.1, an NK cell marker, and an invariant antigen receptor encoded by V␣14 and J␣281 gene segments. 1-3 V␣14 NKT cells recognize a glycolipid, ␣-GalCer, in a CD1d-depenent fashion. 4 The CD1d molecule has been well conserved throughout mammalian evolution and lacks allelic polymorphism. 5-7 After activation, V␣14 NKT cells inhibit tumor metastasis in certain experimental animal models. 8 -10 Furthermore, i.v. injection of ␣-GalCer-pulsed DCs expressed a potent antitumor effect in a B16 melanoma liver metastasis model, where metastasized tumor nodules were eradicated. 11 Human NKT cells bearing the invariant V␣24J␣Q receptor are considered to be the counterpart of murine V␣14 NKT cells and may recognize antigens similar to those of murine V␣14 NKT cells because of striking homology between human V␣24 and mouse V␣14 receptors, particularly in their complementarity-determining region 3. 2 Indeed, human V␣24 NKT cells were activated by ␣-GalCer in a CD1d-dependent fashion 5,12,13 and showed strong antitumor activity upon ␣-GalCer stimulation. 14,15 Human V␣24 NKT cells play crucial roles in various immune responses, including antitumor and autoimmune responses. 16 -19 These reports demonstrated selective reduction in V␣24 NKT cell numbers. Little is known, however, about the nature of V␣24 NKT cells in primary lung cancer patients.We investigated the function of V␣24 NKT cells in peripheral blood and lung of patients with lung cancer. The numbers of V␣24 NKT cells were reduced, whereas the function of freshly isolated V␣24 NKT cells appeared to be preserved in tumor-bearing patients. Furthermore, the ability to present ␣-GalCer by patient DCs was within a normal range. Administration of ␣-GalCer-pulsed DCs expanded V␣14 NKT cells in the lung more than 10 ti...

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