The human telomerase reverse transcriptase (hTERT) is an essential component of the holoenzyme complex that adds telomeric repeats to the ends of chromosomes. The hTERT transcript has been shown to have two deletion type alternative splicing sites. One deletion site induces the alpha-deletion variant, lacking 36 bp from exon 6, and the other induces the beta-deletion variant, lacking 182 bp from exons 7 and 8. Here, we identified a novel deletion variant of the hTERT transcript in hepatocellular carcinoma cell lines. The deleted transcript was characterized by an in-frame deletion of 189 bp, spanning nucleotides 2710 to 2898, corresponding to the complete loss of exon 11 (gamma-deletion). The region lacking in the gamma-deletion lies within RT motifs D and E, suggesting that it is missing conserved residues from the catalytic core of the protein. Both gamma- and alpha-deletion variants were occasionally detected, but the beta-deletion variant was frequently observed. Our results may provide important information for more detailed studies on the regulation of telomerase activity.

The N-terminal amino acid sequence of TA02 (molecular weight 35.0 kDa, isoelectric point 5.29), which is associated with primary lung adenocarcinoma, was determined and a fragment peptide was used to generate mouse monoclonal antibodies (mAbs) against TA02. The amino acid sequence suggested that TA02 might be homologous with napsin A, a new type of aspartic proteinase. In this context, we confirmed the expression of napsin A in primary lung adenocarcinoma using reversetranscription polymerare chain reaction (RT-PCR) and showed that the TA02 mAbs reacted with glutathione-S-transferase (GST)-napsin A fusion protein. We concluded that TA02 is the same molecule as napsin A, and showed immunohistochemically that it is distributed mainly in type II pneumocytes, alveolar macrophages, renal tubules and exocrine glands and ducts in the pancreas. In particular, type II pneumocytes and alveolar macrophages showed high expression of TA02 among human normal tissues. In primary lung adenocarcinoma, 47 out of 58 (81.0%) primary lesions were positive. All well-differentiated adenocarcinomas except those of goblet cell type showed high expression of TA02. In addition, two out of seven (28.6%) large cell carcinomas showed low expression of TA02. The other histopathological types of primary lung cancer did not express TA02 at all. A few cases of renal cell cancer, pancreatic cancer, breast cancer, thyroid cancer, colon cancer and ovarian cancer showed low expression, but the staining patterns were completely different from that of primary lung adenocarcinoma, which showed a granular staining pattern. Our novel mAbs should be valuable for immunochemical detection of TA02/napsin A.Key words: TA02 -napsin A -monoclonal antibody against TA02 -human aspartic proteinaseprimary lung adenocarcinoma Primary lung carcinoma incidence and mortality are still increasing. Even though primary lung carcinoma originates in bronchial epithelium, it shows a wide range of histopathological patterns. Adenocarcinoma accounts for approximately 40% of primary lung carcinoma in Japan. Most adenocarcinomas appear in peripheral bronchi. It is thought that this type of tumor originates from type II pneumocytes of the alveolar sac and Clara cells of the bronchiole. Other types of adenocarcinoma, which occur in relatively large bronchi, seem to derive from mucus cells, duct epithelial cells, goblet cells or bronchial glands. Based on the suspected cells of origin, primary lung adenocarcinoma can be subdivided into four types: type II pneumocyte type, Clara cell type, goblet cell type and bronchial gland type. 1)We have already reported the specific two-dimensional polyacrylamide gel electrophoresis patterns of each histological type of primary lung carcinoma.2) In that investigation we discovered TA02 polypeptide (TA02; molecular weight 35.0 kDa, isoelectric point 5.29), which is associated with primary lung adenocarcinoma.3) The intensity of TA02 expression reflected the degree of histopathological differentiation of primary lung adenocarcinoma, except for th...

Ubiquitin, which can conjugate with cellular proteins, is classified into two forms: free ubiquitin and multiubiquitin chains. The latter is active as a signal for degradation of the targeted proteins. We found both forms in human serum and, using two immunoassays, quantitated them in sera from healthy subjects and patients with some diseases. Because of putative leakage of erythrocyte ubiquitin, hemolytic serum and serum obtained after long incubation (>1–2 h) of blood at room temperature were excluded. Serum concentrations of multiubiquitin chains and free ubiquitin were substantially higher in rheumatoid arthritis and hemodialysis patients, respectively, than healthy subjects. Additionally, in acute viral hepatitis, serum multiubiquitin chain concentrations were increased in the acute phase, decreased in the recovery phase, and correlated with alanine and aspartate aminotransferase activities (r = 0.676 and 0.610, P <0.0001 and <0.001, respectively). Therefore, serum ubiquitin may have prognostic value.

Daunomycin was covalently attached via a dextran bridge to specific antibodies against rat a-fetoprotein produced in a horse. The effect of this conjugate on an ei-fetoproteinproducing tumor was investigated in terms of cytotoxicity and inhibition or retardation of tumor development. Under the experimental conditions used, the covalent conjugate was by both criteria more efficient than either daunomycin alone or a mixture of daunomycin and specific antibodies or a conjugate ofdaunomycin with horse immunoglobulin. These results show that the conjugate may be useful as a specific cytotoxic agent against at-fetoprotein-producing tumors.The cytotoxic activity of a specific antiserum to a-fetoprotein (AFP) on AFP-producing tumors was determined both in vitro (1, 2, t) and in vivo (3, 4). The observation that tumor cells that produce very low levels of AFP are able to survive selectively on treatment with the antiserum in vitro (5) suggests that the cytotoxic activity of the antiserum may depend on the level of AFP production by the tumor cells. Recent studies (1, 6, 7) have shown that AFP is detectable on the cell surface of AFP-producing tumor cells and suggest that the binding of antibody to AFP may have a fundamental role in its cytotoxic effect on such cells.Daunomycin (8, 9) is a well-known antitumor drug, widely used in cancer therapy. The major drawback, however, is that daunomycin, like many other drugs effective in killing tumor cells, also has detrimental effects on rapidly proliferating normal cells. This toxicity, which limits the effective use of chemotherapy in treatment of neoplastic diseases, may perhaps be overcome or reduced by binding the drug to a carrier that has specific affinity to the tumor cells (10,11). In previous studies, we have tested the effects of daunomycin-antitumor immunoglobulin conjugates on various tumors (12). These conjugates retained most of their original drug and antibody activities (13,14). Conjugates with specific antibodies were more effective than drug conjugates with normal immunoglobulin and, under certain conditions, were an improvement over the free drug (15).The aim ofthis study was to test specific antibodies produced in a horse against rat AFP as carriers of daunomycin. Daunomycin-anti-AFP conjugates were tested for their effect on rat hepatoma and compared with free daunomycin, anti-AFP, and a mixture of anti-AFP and daunomycin linked to normal horse immunoglobulin. A preliminary report ofthese studies has been presented. § A report describing the in vitro effect of daunomycin attached by a direct linkage to anti-mouse a-fetoprotein has also appeared (16). MATERIALS AND METHODSSpecific Antibodies to AFP. Specific antiserum to rat AFP was produced in a horse by weekly subcutaneous injections of 1 mg of purified AFP (17) emulsified in Freund's complete adjuvant. Specific antibodies to rat AFP were purified by affinity chromatography on activated Sepharose 4B coupled to rat AFP (18). (Fab')2 fragment from the specific antibody was prepared by pepsin digesti...

A sandwich ELISA has been developed to measure intracellular levels of multi-ubiquitin chains. The mixture of multi-ubiquitin chains, prepared in vitro by incubation of ubiquitin (plus "'I-ubiquitin) and lysozyme with ubiquitin-ligating enzymes and ATP, was partially purified and established as a standard named the multi-ubiquitin-chain reference preparation 1 (MUCRPI). The concentration of MUCRPI was calculated from the recovered radioactivity of "'I-ubiquitin. All measurements by the ELISA were expressed in terms of MUCRPI. The ELISA showed good sensitivity (98 pg/ml), precision (intra-assays < 6%) and reproducibility (interassay < 9%). In addition, there was no substantial cross-reaction with mono-, di-and tri-ubiquitin, or mono-ubiquitinated and di-ubiquitinated lysozyme in the ELISA, and large multi-ubiquitin chains ( n > approximately 6 ) may be fully reactive. These results combined with excellent results in the recovery and dilution tests guarantee accurate measurement of multi-ubiquitin chains in cell extracts prepared with a lysis buffer (water soluble) or the buffer supplemented 8 M urea (urea soluble). The level of the water-soluble multi-ubiquitin chains in reticulocytes was lower than that of erythrocytes, but the urea-soluble chain level was higher in the reticulocytes. Heat-shock treatment of HeLa cells increased the urea-soluble multi-ubiquitin chains. These data indicate that this ELISA provides a useful and reliable approach to the study of intracellular multi-ubiquitin-conjugate turnover.Keywords: ELISA ; heat shock ; multi-ubiquitin chain; reticulocyte; ubiquitin.Ubiquitin is found in either a free form or a form bound covalently to intracellular proteins in vivo (Hershko, 1988;Finley and Chau, 1991). In the latter form, the ubiquitin C-terminus is ligated to &-amino groups of appropriate Lys residues in target proteins by a family of ubiquitin-conjugating enzymes: a ubiquitin-activating enzyme (El), a ubiquitin-carrier protein (E2), and a ubiquitin-protein ligase (E3) (Hershko et al., 1983;Hershko and Ciechanover, 1992). In this process, several ubiquitin moieties are usually ligated sequentially to the initial acceptor protein, forming multi-ubiquitin chains in which the ubiquitin Cterminus is conjugated to the &-amino group of Lys48 of another ubiquitin moiety (Chau et al., 1989). The multi-ubiquitin chain acts as a signal to induce degradation of target proteins by 2 6 s proteasome (Chau et al., 1989;Hershko and Ciechanover, 1992). Therefore, the chain structure is essential for ubiquitin-dependent proteolysis.The ubiquitin-dependent proteolysis has been implicated in a variety of cellular processes, including stress response (Finley et al., 1987;Carlson et a]., 1987), cell-cycle control (Glotzer et al., 1991;Nishizawa et al., 1993) and apoptosis (Delic et al., 1993). Intracellular levels of ubiquitin and ubiquitin-protein conjugates are changed in response to heat shock (Parag et al., Abbreviations. MUCRPI , multi-ubiquitin chain reference preparation 1 ; E l , ubiquitin-activating enzy...