Search PubMed⌕ Search

Biomedical subjects

S Sakiyama

Publications and source records attributed to S Sakiyama.

At least 163 records · Page 9Linked to original sources
PubMed1989

Amounts and modulation of actin mRNAs in mouse oocytes and embryos.

In order to measure the content of beta- and gamma-actin mRNA in mouse oocytes and ovulated eggs, Northern and slot blots were hybridized to complementary RNA probes transcribed from mouse isotype-specific cDNA sequences. The blots included samples of isotype-specific sense strand RNA standards prepared from the same cDNA sequences. Total actin mRNA content was estimated to be 40 fg per preovulatory full-grown oocyte or egg, consisting of one-third beta-actin mRNA and two-thirds gamma-actin mRNA. Ninety per cent of the actin mRNA is on polysomes in full-grown oocytes. The per cent of actin mRNA in polysomal mRNA is similar to the per cent of actin in newly synthesized proteins. Measurements on other developmental stages showed that, in mid-growth-phase oocytes, each actin mRNA reaches a level twofold higher than in full-grown oocytes. Thereafter, all modulations of the two isotypic mRNAs occur in parallel; that is, they are maintained at constant levels during the late growth phase (oocytes from females 8-14 days old); gradually degraded in oocytes that have completed their rapid growth phase (oocytes from females 15-18 days old), in maturing oocytes, and in 1- and 2-cell embryos; and deadenylated after about 7 h of progression into meiotic maturation.

Actins↗
PubMed1989

Cysteine proteinase-inhibitory activity of ras gene product is not affected by mutations at GTPase-activating protein-binding sites.

We have investigated the proteinase-inhibitory activity against cathepsin L of some c-Ha-ras gene products (p21s) with point mutations at position 12, 33 or 35, which affect the interaction with GTPase-activating protein (GAP). All of the full-length p21s examined showed similar inhibitory activities irrespective of the point mutation and the transforming activity. These results suggest that mutations at GAP-interacting sites have no effect on the proteinase-inhibitory activity of p21s and that the proteinase-inhibitory activity alone is not sufficient for the transformation caused by p21s.

Amino Acid Sequence↗
PubMed1989

Potentiation of cysteine proteinase-inhibitor activity of full-length Ha-ras oncogene products by denaturation-renaturation.

We have investigated protease-inhibitory activity against cathepsin L of human and murine Ha-ras oncogene products (p21s) produced by Escherichia coli. The inhibitory activity of full-length p21s was much weaker than that of truncated p21s, however, the inhibitory activity was potentiated after denaturation with 8 M urea and 2 M NaCl followed by renaturation. These suggest that p21 can be folded into multiple three-dimensional conformations which have different protease-inhibitory activities.

Animals↗
PubMed1988

Suppression of synthesis of tropomyosin isoform 2 in metastatic v-Ha-ras-transformed NIH3T3 cells.

We have reported that the synthesis of tropomyosin isoform 2 (TM2) is significantly suppressed in high-metastatic Lewis lung carcinoma cells compared with that in low-metastatic cells. In order to examine whether the change is also observed in other high-metastatic tumor cells, we compared the pattern of expressions of TM isoforms between NIH3T3 cells and v-Ha-ras-transformed NIH3T3 (pH1-3) cells, the latter of which was highly metastatic when injected into BALB/c nude mice. The results showed that the expression of TM2 was less in pH1-3 cells than in NIH3T3 cells, suggesting that the suppression of TM2 synthesis is involved in the expression of metastatic phenotype.

Animals↗
PubMed1988

Isolation and characterization of a complementary DNA clone for a Mr 32,000 protein which is induced with tumor promoters in BALB/c 3T3 cells.

The synthesis of a protein of Mr 32,000 (p32) is enhanced by various tumor promoters, chemical carcinogens, metal salts, and heat shock in BALB/c 3T3 cells. We have isolated a complementary DNA (cDNA) clone for p32 from a lambda gt10 library of BALB/c 3T3 cells. The library was constructed from mRNA extracted from the cells treated with sodium arsenite, which stimulates the p32 expression most effectively among various agents so far tested. Having screened this library differentially with probes which represent induced and uninduced mRNA populations for p32, we first obtained a partial p32 cDNA clone and have subsequently succeeded in the isolation of a cDNA clone containing the entire coding sequence. RNA blot analysis has shown that p32 mRNA is induced as early as 0.5 h after the addition of 12-O-tetradecanoyl-phorbol-13-acetate or sodium arsenite. Computer-assisted comparison with GenBank data has revealed a striking similarity in the nucleotide sequences between cDNAs of p32 and rat heme oxygenase. These results strongly suggest that p32 is a mouse homolog of this enzyme.

Animals↗
PubMed1988

Degradation of epidermal growth factor receptors by cathepsin L-like protease: inhibition of the degradation by c-Ha-ras gene products.

Extract of NIH3T3 mouse fibroblasts contains a protease which can cleave epidermal growth factor receptor (EGF receptor). This protease was tentatively named cathepsin X and purified to near homogeneity. The characteristics of cathepsin X were similar to those of cathepsin L and the proteolytic activity of cathepsin X was inhibited by c-Ha-ras gene products.

Animals↗
PubMed1988

Inhibition of cathepsin L-induced degradation of epidermal growth factor receptors by c-Ha-ras gene products.

The inhibitory activities of c-Ha-ras gene products (p21s) toward several cysteine proteinases have been investigated. The activity of cathepsin L was inhibited by p21s most effectively while those of cathepsin B and papain were slightly inhibited by p21s. p21s did not show any inhibitory activity toward cathepsin H. In order to connect the protease-inhibitor activity of p21s with cell growth, the degradation of epidermal growth factor receptors (EGF-receptors) was investigated. EGF-receptors were preferentially cleaved by cathepsin L but not by cathepsin B or H. The cleavage of EGF-receptors by cathepsin L was inhibited by p21s dose-dependently. These results raise the possibility that p21s can suppress the degradation of growth-related proteins such as EGF-receptors and thereby affect cell growth.

Carcinoma, Squamous Cell↗
PubMed1988

Isolation and characterization of a cDNA that encodes mouse fibroblast tropomyosin isoform 2.

We isolated and characterized a cDNA clone encoding tropomyosin isoform 2 (TM2) from a mouse fibroblast cDNA library. TM2 was found to contain 284 amino acids and was closely related to the rat smooth and skeletal muscle alpha-TMs and the human fibroblast TM3. The amino acid sequence of TM2 showed a nearly complete match with that of human fibroblast TM3 except for the region from amino acids 189 to 213, the sequence of which was identical to those of rat smooth and skeletal muscle alpha-TMs. These results suggest that TM2 is expressed from the same gene that encodes the smooth muscle alpha-TM, the skeletal muscle alpha-TM, and TM3 via an alternative RNA-splicing mechanism. Comparison of the expression of TM2 mRNA in low-metastatic Lewis lung carcinoma P29 cells and high-metastatic D6 cells revealed that it was significantly less in D6 cells than in P29 cells, supporting our previous observations (K. Takenaga, Y. Nakamura, and S. Sakiyama, Mol. Cell. Biol. 8:3934-3937, 1988) at the protein level.

Amino Acid Sequence↗
PubMed1988

Isolation of cDNA clones for mouse cytoskeletal gamma-actin and differential expression of cytoskeletal actin mRNAs in mouse cells.

We described the structures of mouse cytoskeletal gamma-actin cDNA clones and showed that there is strong conservation of the untranslated regions with human gamma-actin cDNA. In addition, we found that the expression levels of beta- and gamma-actin mRNAs are differentially controlled in various mouse tissues and cell types but are coordinately increased in the cellular growing state. These results suggest that there are multiple regulatory mechanisms of cytoskeletal actin genes and are consistent with the argument that beta- and gamma-actins might have functional diversity in mammalian cells.

Actins↗
PubMed1988

Differential expression of a tropomyosin isoform in low- and high-metastatic Lewis lung carcinoma cells.

Two-dimensional electrophoretograms of newly synthesized polypeptides from low-metastatic (P29) and high-metastatic (D6) Lewis lung carcinoma cells were compared. The results showed that the synthesis of tropomyosin 2 (TM2) was significantly less in D6 cells than in P29 cells. Furthermore, suppression of TM2 synthesis was induced in P29 cells during incubation in medium containing dimethyl sulfoxide or butyric acid, which induced the metastatic phenotype of P29 cells. These results suggest that the suppression of TM2 synthesis is linked to the metastatic potential of Lewis lung carcinoma cells.

Animals↗
PubMed1987

Enhanced expression of a glyceraldehyde-3-phosphate dehydrogenase gene in human lung cancers.

We have shown that a Mr 37,000 protein whose expression is strongly enhanced in human lung cancer tissues is the subunit of glyceraldehyde-3-phosphate (GAPDH, EC 1.2.1.12). We have isolated a GAPDH complementary DNA from human lung cancer cells and deduced the complete amino acid sequence of the encoded protein. The structure of the lung cancer GAPDH is identical to that of liver GAPDH. In addition, we have found that GAPDH mRNA expression is markedly increased in human lung cancer tissues. These results disclose a molecular basis of increased glycolysis in cancer cells and reveal an important role of energy creating reaction in cancer cell growth.

DNA↗
PubMed1987

Degradation of a cAMP-binding protein is inhibited by human c-Ha-ras gene products.

Incubation of the particulate fraction of cell extract prepared from NIH3T3 mouse fibroblasts resulted in preferential proteolytic degradation of a cAMP-binding protein. The proteolysis was inhibited by human c-Ha-ras gene products produced by Escherichia coli. The proteolysis was observed at pH 6 to 7, and inhibited by antipain and leupeptin. These results suggest that cAMP-binding proteins might be cleaved by thiol proteinases. In fact, c-Ha-ras gene products were proved to inhibit the cathepsin B-like activity present in the particulate fraction.

Animals↗
PubMed1987

Defects of cyclic adenosine 3':5'-monophosphate-dependent protein kinases in initiated clones derived from BALB/c 3T3 mouse fibroblasts.

Two-stage carcinogenesis is involved in the transformation of mouse fibroblasts BALB/c 3T3 cells. In order to investigate the role of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase at the stage of initiation, the following experiments were carried out: (a) two initiated clones (M14, M20) which exhibit 12-O-tetradecanoylphorbol-13-acetate-dependent growth in soft agar medium were isolated from cells treated with N-methyl-N'-nitro-N-nitrosoguanidine. The activity of cAMP-dependent protein kinase in M14 was reduced while that in M20 was similar to the level in parental cells. However, cAMP-binding activity to a regulatory subunit of cAMP-resistant clones were isolated from 4-nitroquinoline oxide- or ethyl methanesulfonate-treated cells. These clones have reduced activities in both cAMP-binding and cAMP-dependent protein kinase itself. Two of three cAMP-resistant clones were proved to be able to grow in soft agar medium only in the presence of 12-O-tetradecanoylphorbol-13-acetate.

4-Nitroquinoline-1-oxide↗
PubMed1987

c-Ha-ras gene products are potent inhibitors of cathepsins B and L.

c-Ha-ras proteins produced by Escherichia coli inhibited the activities of cathepsins B and L which had been partially purified from rat kidney. Furthermore, amino acid sequence homology between c-Ha-ras proteins and thiol proteinase inhibitors has been found.

Amino Acid Sequence↗
PubMed1986

Altered properties of cAMP-dependent protein kinase in H-ras-transformed NIH3T3 cells.

cAMP-dependent protein kinase activity in the soluble fraction was decreased in both v-H-ras-transformed and activated-c-H-ras-transformed NIH3T3 cells as compared with that in NIH3T3 cells. Both of the elution profile of type II cAMP-dependent protein kinase from DEAE-cellulose and the electrophoretic behavior of its regulatory subunits in the particulate fraction of H-ras-transformed cells are different from those of control NIH3T3 cells. These results suggest that ras protein causes the alterations of some properties of cAMP-dependent protein kinases.

Animals↗
PubMed1986

Enhanced expression of a 37,000-dalton protein in human lung cancers.

When proteins in the tissue extracts of lung cancers were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), a 37,000-dalton protein (37K) was detected as a prominent form in all cases thus far examined. This protein was barely detectable or its amount was very small in the normal lung tissue extracts. Two-dimensional PAGE of lung tumor extracts showed 37K as a major spot with an isoelectric point of 7.6. An antibody against 37K was raised in a rabbit by injecting purified 37K. Immunoblot analysis of normal lung and lung tumor tissues revealed that the affinity-purified anti-37K antibody specifically recognized 37K and that the intensities of immunostaining corresponded to those of Coomassie brilliant blue staining.

Electrophoresis, Polyacrylamide Gel↗
PubMed1986

Similarity between glyceraldehyde-3-phosphate dehydrogenase and a 37,000-dalton protein which is abundantly expressed in human lung cancers.

Human lung cancers of all histological types contain a protein of 37,000 daltons (37K) as an abundant component. Partial sequence analysis of purified 37K revealed a strong homology with glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12). Tryptic peptide mapping analysis showed that the pattern of 37K was very similar to those of GAPDHs both purified from lung tumor and obtained commercially. An antibody raised against 37K in a rabbit also reacted with authentic GAPDH. These results suggest a possible involvement of GAPDH itself or a GAPDH-related protein in lung tumorigenesis.

Amino Acid Sequence↗