Nozomu Hibi

Expression profile of a γ-deletion variant of the human telomerase reverse transcriptase gene

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.

Carbonic anhydrase-III immunohistochemical localization in human skeletal muscle

SummaryUsing the indirect immunoperoxidase method, we studied the localization of carbonic anhydrase-III (CA-III) in frozen sections of biopsies of human skeletal muscle which had no definite pathology. CA-III was found to be localized in Type-I muscle fibers when compared with serial sections stained with myosin ATPase and other reactions. Our finding was in accordance with the biochemical data so far reported. It was thought that CA-III could be used as a marker for abnormal Type-I muscle fibers in several neuromuscular diseases.

Ability of ubiquitin radioimmunoassay to discriminate between monoubiquitin and multi-ubiquitin chains

Free ubiquitin (mainly monoubiquitin) and multi-ubiquitin chains coexist in eukaryote cells and serve distinct cellular roles. However, any immunoassay systems established previously have not been proved to be applicable for measuring the former without cross-reactive responses with the latter. For this purpose, we developed a radioimmunoassay specific to monoubiquitin by employing antiserum US-1 against ubiquitin. In this assay, ubiquitin-protein conjugates, prepared by a reticulocyte lysate fraction II and fractionated on Moro Q and Superdex 200 columns, exhibited practically no cross-reactivity. The cross-reactivity of fractionated ubiquitin-lysozyme conjugates was also analyzed as a function of their multi-ubiquitin chain size. As a result, the larger the conjugates were found to be, the weaker were the cross-reactive responses they showed, and the multi-ubiquitin chains (n > approx. 20) were substantially unreactive in the radioimmunoassay. By using the radioimmunoassay, heat-shock-induced decrease in the level of cellular free (mono)ubiquitin was detected. In addition, the standard preparation of multi-ubiquitin chains was not cross-reactive in all other five radioimmunoassays employing distinct antibodies to ubiquitin (four antisera and a monoclonal antibody). These data suggest that radioimmunoassays employing ubiquitin antibodies raised by the general methods can discriminate between monoubiquitin and multi-ubiquitin chains and quantitate cellular free ubiquitin.

Pleiotropic phenotypic expression in cybrids derived from mouse teratocarcinoma cells fused with rat myoblast cytoplasts

Cybrid clones were isolated by fusing mouse embryonal carcinoma (PCC4) cells with cytoplasts of rat myoblastic cells (L6TG X CAPr). Although some clones were similar to PCC4 (Type II), a high proportion (88%) were differentiated; the differentiated cells had a mesh-like arrangement (Type I) or were flat with many projections (Type III). Protein patterns of both Type I and Type III cells changed markedly from that of PCC4 cells. Type III cells lacked alkaline phosphatase and expressed endo A and B proteins predominantly. One Type III clone produced alpha-fetoprotein and plasminogen activator (visceral endoderm-like), while another clone consisted of trophectodermal cell-like giant cells. Therefore it was shown that introduction of the somatic cell cytoplasm induces differentiation of teratocarcinoma stem cells, suggesting a cytoplasmic element (or elements) regulating gene expression.

Selective in vitro and in vivo growth inhibition against human yolk sac tumor cell lines by purified antibody against human α-fetoprotein conjugated with mitomycin C via human serum albumin

SummaryThe anticancer drug mitomycin C (MMC) was conjugated with an affinity-purified horse antibody to human α-fetoprotein (aAFP) with human serum albumin (HSA) as the intermediate drug carrier. The conjugate (aAFP:HSA:MMC molar ratio, 1:1:30) retained full antibody binding activity as determined by a competitive binding radioimmunoassay. In a cytotoxicity test in which the AFP-producing human yolk sac tumor TG-1 cells were preincubated with test materials for 2 h followed by an additional 48-h culture in fresh medium, the conjugate was 20-fold more cytotoxic than free MMC at an equivalent MMC concentration of 100 ng/ml. The in vivo antitumor effect of the conjugate was tested against the human yolk sac tumor JOG-9 growing in athymic nude mice. When the tumor-bearing mice were treated with a total of 6 injections given on 2 consecutive days and then every other day starting 8 days after SC tumor inoculation [2 (equivalent MMC) μg/head per injection], the conjugate retarded tumor growth more effectively than free MMC and normal horse immunoglobulin conjugate.

Development of a highly sensitive enzyme-immunoassay for serum carbonic anhydrase-III

A highly sensitive sandwich enzyme-immunoassay (EIA) for human muscle carbonic anhydrase isozyme III (CA-III) has been developed using microplate as a solid-phase and peroxidase as a labelled enzyme. The assay can detect levels as low as 2 ng/ml when 20 microliter of sample sera were used. Sera from patients with various neurological diseases were studied using this method, and elevated serum CA-III levels were found in patients with Duchenne muscular dystrophy, limb-girdle dystrophy, fascioscapulohumeral dystrophy, polymyositis and amyotrophic lateral sclerosis. The values correlated well with the results of radioimmunoassay (RIA), with a correlation coefficient of 0.92 (P less than 0.001). We feel EIA is preferable to RIA for its simple methodology.

Main serum protein of rainbow trout (Salmo gairdnerii): its biological properties and significance

Abstract 1. 1. Main serum α 1 -protein (α 1 P) of rainbow trout was purified and its biochemical and physico-pathological properties were studied. 2. 2. α 1 P was suggested to be a primitive protein having both properties of albumin and AFP in serum proteins of mammals according to the following results. 3. 3. Molecular weight (75,000), two kinds of molecules (pI 4.55 and 5.05) and amino acid composition. 4. 4. Dye- or ConA binding activity. 5. 5. Estrogen binding activity and inhibitory effect on lymphoblastoid-forming activity. 6. 6. Possible osmotic regulator. 7. 7. Significant elevation of blood α 1 P level in the course of hepatoma induction.

Evaluation of a conjugate of purified antibodies against human AFP-dextran-daunorubicin to human AFP-producing yolk sac tumor cell lines

SummaryThe anticancer drug, DNR, was conjugated to an affinity-purified horse antibody to human AFP (aAFP) via a dextran bridge. The conjugate (immunoglobulin: DNR molar ratio, 1:50) was twice as potent as free DNR in an in vitro cytotoxicity assay against an AFP-producing human yolk sac tumor. The in vivo effect of aAFP, DNR, and the conjugate was tested against the human yolk sac tumor growing in nude mice. The conjugate, at a concentration of DNR containing the equivalent amount of 20 μg or 70 μg/mouse significantly retarded tumor growth whereas free aAFP showed only a slight inhibition of tumor growth compared to the PBS-treated control. Mice which received 20 μg/mouse of free DNR showed a moderate retardation of tumor growth whereas those which received 70 μg/mouse of DNR or a mixture of DNR and aAFP showed emaciation and early death due to acute toxicity of the drug. These results suggest that the anti-body-drug conjugate accumulated preferentially on the AFP-producing tumor cells and that cytotoxicity occurred.

Calpain inhibitor inhibits secretory granule maturation and secretion of GH.

Clathrin- and AP-1-coated buds are present on immature secretory granules of endocrine cells that mature into clathrin-uncoated granules. The mechanism of clathrin and adaptor protein uncoating has remained obscure. Benzyloxycarbonyl-L-leucyl-L-leucinal (ZLLal), a calpain inhibitor, reduced growth hormone (GH) secretion with intracellular accumulation, in a GH-secreting rat pituitary tumor cell. Pulse and chase demonstrated that ZLLal retarded the turnover of clathrin (Clt.H) and adaptins. ZLLal-treatment co-immunoprecipitated the increased amounts of GH with Clt.H and adaptins compared to control cells, suggesting the intracellular accumulation of immature secretory granules. Clt.H and adaptins were limited-proteolyzed by m-calpain in vitro, indicating that calpain may be involved partly in the maturation of secretory granules in endocrine cells via the process of clathrin uncoating.

Purification of specific antibody to α-foetoprotein and its immunological effect on cancer cells

Abstract Human or rat α-foetoprotein (AFP) was highly purified from ascitic fluid or serum of hepatoma bearers. The purification was carried out mainly by means of immunoadsorbent chromatography using Sepharose coupled to specific anti-AFP antibody with BrCN activation, and by Sephadex gel filtration. Horses were immunized with the purified AFP and the specific antibody was isolated from the antiserum by means of an immunoadsorbent coupled to purified AFP. The specific antibody was found to bind specifically with AFP-producing tumour cells. The antibody was applied for radio immunodetection of the tumour. 125 I-labelled antibody was administered to patients or rats with hepatoma, and radioactivity localized in the tumours was scintiscanned with a scintillation camera. In this way, the location of the tumour was detected in about 50% of the hepatoma bearers. The cytotoxicity of the antibody was clearly demonstrated both vitro and in vivo in animal experiments. The antibody was administered to patients with advanced hepatoma. Although no improvement of the disease was demonstrated, serum AFP levels decreased greatly and in some cases low AFP levels were maintained for long periods suggesting that the antibody suppressed AFP-producing hepatoma cells. No significant side effects were observed in patients who had been administered with the horse antibody.