Journal Article Myeloid Calcium Binding Proteins: Expression in the Differentiated HL-60 Cells and Detection in Sera of Patients with Connective Tissue Diseases Get access Ryoko Kuruto, Ryoko Kuruto *Laboratories of Microbiology University of Shizuoka School of Food and Nutritional ScienceYada, Shizuoka 422 Search for other works by this author on: Oxford Academic PubMed Google Scholar Ryushi Nozawa, Ryushi Nozawa *Laboratories of Microbiology University of Shizuoka School of Food and Nutritional ScienceYada, Shizuoka 422 Search for other works by this author on: Oxford Academic PubMed Google Scholar Keiichi Takeishi, Keiichi Takeishi **Laboratories of Genetic Engineering, University of Shizuoka School of Food and Nutritional ScienceYada, Shizuoka 422 Search for other works by this author on: Oxford Academic PubMed Google Scholar Kyoko Arai, Kyoko Arai ***Departments of Bacteriology Juntendo University School of MedicineBunkyo-ku, Tokyo 113 Search for other works by this author on: Oxford Academic PubMed Google Scholar Takeshi Yokota, Takeshi Yokota ***Departments of Bacteriology Juntendo University School of MedicineBunkyo-ku, Tokyo 113 Search for other works by this author on: Oxford Academic PubMed Google Scholar Yoshinari Takasaki Yoshinari Takasaki ****Departments of Medicine, Juntendo University School of MedicineBunkyo-ku, Tokyo 113 Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 108, Issue 4, October 1990, Pages 650–653, https://doi.org/10.1093/oxfordjournals.jbchem.a123257 Published: 01 October 1990 Article history Received: 23 May 1990 Published: 01 October 1990
(−)-Epigallocatechin gallate (EGCG), a major component of green tea catechins, is known to inhibit cell growth and to induce apoptosis in a variety of cultured cells. We examined effects of green tea catechins in cultured cells derived from human gastric carcinoma. The proliferation of four cell lines (MKN-1, MKN-45, MKN-74 and KATO-III) was inhibited with EGCG in a dose-dependent manner. The growth of MKN-45 cells was most efficiently inhibited by the treatment (IC50: 40 μM EGCG) among the four cell lines, while KATO-III cells were most insensitive (IC50: 80—150 μM) to the EGCG treatment. In addition, (−)-epicatechin (EC) had a major synergistic effect on the induction of apoptosis in MKN-45 cells treated with EGCG; however it had little effect on the inhibition of cell growth induced by EGCG. To study the molecular mechanisms behind the induction of apoptosis by EGCG, the activity of caspases in MKN-45 cells treated with EGCG was examined. Activity levels of caspases-3, -8 and -9 were elevated in EGCG-treated cells, suggesting that these caspases are involved in the apoptosis induced by EGCG. Furthermore, the synergistic effect of EC with EGCG on the induction of apoptosis was specifically canceled by catalase treatment, suggesting that the synergism involves the extracellular production of reactive oxygen species.
To identify the essential motifs of the promoter of the human thymidylate synthase (hTS) gene, we constructed a set of deletion mutants that covers the region from -441 to +28 of the hTS gene. (The nucleotide positions are numbered from the first position of the initiation codon of the hTS gene.) From the results of chloramphenicol acetyltransferase (CAT) assay of these mutants, two positive elements for the promoter activity were identified: one contains CACCC box (CCACACCC) that is found in the SV40 enhancer motif and the other contains the sequence that is homologous to the Sp1 binding site in the mouse TS gene. Furthermore, two negative regulatory sequences were identified between the two positive elements and upstream from the CACCC box. Cassette mutations were introduced into these motifs and the function of the motifs was confirmed. From the results of gel mobility shift analysis, we found that three nucleoprotein complexes were formed in the promoter region of the hTS gene. The formation of one of the complexes was competed by the DNA fragment bearing the GC box. The gel mobility shift analyses using the DNA fragments with cassette mutations revealed that the complex was formed on the Sp1 binding site of the hTS gene and the formation of the complex correlated with the promoter activity of the fragments with cassette mutations measured by the CAT assay.
Low molecular weight RNAs were isolated from nuclei of the cellular slime mold Dictyostelium discoideum AX-3. Analysis of the RNAs by polyacrylamide gel electrophoresis showed that the vegetative cell nuclei contained, besides tRNA (Ddl), 5S RNA (Dd4), and 5.8S RNA (Dd7), at least 7 small RNA species (Dd3, Dd5, Dd6, Dd8, Dd9, DdlO, Ddll) of 4S to 8S as major components and that the 7 small RNAs were localized mainly in the nucleus and had no poly(A) sequence. These nuclear RNA species were metabolically stable, as shown by a chase experiment. Dd6, Dd8, and Dd9 had similar gel electrophoretic mobilities to those of the small nuclear RNA species Ul, U2, and U3, respectively, of rat liver. Analysis of the 5′-terminus of these RNA molecules with tobacco acid phosphodiesterase suggested that Dd6, Dd8, and Dd9 each contain a cap. Sequence analysis of the 3′-end labeled Dd9 RNA showed that the 3′-terminal region sequenced had sequence homology with that of rat Novikoff hepatoma U3 RNA. These results indicate that Dictyostelium nuclei contain a set of small nuclear RNA species which is structurally similar to that in mammalian cells. No qualitative differences were detected between the small nuclear RNA species of vegetative and early differentiating cells.
Human thymidylate synthase [EC 2.1.1.45] was purified to homogeneity and its NH2-terminal amino acid sequence was determined taking advantage of the following facts: i) The source of the enzyme was a transformant of mouse FM3A mutant cells which lacks mouse thymidylate synthase but overproduces human thymidylate synthase. ii) The enzyme could be purified on two kinds of affinity column, Cibacron blue dye-bound agarose and methotrexate-bound Sepharose. iii) The enzyme could finally be separated from a trace of impurities by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate. The purified human thymidylate synthase had a subunit with a molecular weight of 33,000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was subjected to Edman degradation and the NH2-terminal 24 amino acids were sequenced by successive use of a high-sensitivity gas-phase protein sequencer and high performance liquid chromatography to be as follows:
Thymidylate synthase (TS) catalyzes the conversion of deoxyuridylate to thymidylate. Promoter regions of the TS gene from three vertebrates, human, mouse and rat, have been analyzed so far. A unique inverted repeat was present in the promoter region of the human, but not in the mouse and rat, TS genes, and the feature of the promoter region is considerably different between the human TS gene and the two rodent TS genes. To examine whether the characteristics of the human TS gene promoter is intrinsic to human or primate, we cloned a DNA fragment containing the promoter region of the monkey TS gene and determined the nucleotide sequence in this region. The nucleotide sequence in the promoter region of the moneky TS gene was revealed to have 88% homology with that in the corresponding region of the human TS gene. The unique repeted structure mentioned above was also found in the monkey TS gene. Furthermore, the Sp1-binding motif and the CACCC box were found to be conserved in the monkey TS gene. The result of chloramphenicol acetyltransferase assay suggested that the Sp1-binding motif and the CACCC box are essential for the promoter activity of the monkey TS gene, as in the case of the human TS gene.
Thymidylate synthase-negative mutants of mouse FM3A cells were transformed to thymidine prototrophs by human DNA. The stable transformants had only human thymidylate synthase and segments of human DNA. They grew normally but had unusually high levels of the human enzyme. In two transformants examined, however, neither was the dTTP pool elevated nor the dCTP pool decreased. DNA synthesis in permeabilized cells of a transformant was more efficient than that in the wild type with dATP, dGTP, dCTP, and dUMP as substrates, but this was not so when dUMP was replaced by dTTP. Unlike the mouse enzyme, the human enzyme in the transformants did not co-sediment with DNA polymerase alpha and thymidine kinase in a sucrose gradient, suggesting that the human enzyme is not incorporated into a multienzyme complex for DNA replication. The high levels of the human enzyme in the transformants were suppressed to various degrees by fusion with a wild type mouse line. No active hybrid dimer enzyme was found between the human and mouse enzymes, which each consist of two identical subunits. Thus, the human enzyme in the transformants seems to behave differently from the mouse enzyme and its overproduction seems to be necessary for supporting the normal growth of the transformants.
An unidentified genomic DNA fragment of 2.4kb that is weakly hybridizable with thymidylate synthase (TS) cDNA was cloned from a human genomic DNA library. Sequencing of the cloned DNA fragment and comparison of the sequence with that of the known human TS cDNA revealed that the DNA fragment contained a human TS processed pseudogene with unusual features. Based on the rate of nucleotide substitutions for neutral mutations in the 3'-untranslated regions between the gene and the pseudogene, it was estimated that the human TS pseudogene was formed about 16 million years ago.
