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Biomedical subjects

S Sakiyama

Publications and source records attributed to S Sakiyama.

At least 109 records · Page 6Linked to original sources
PubMed1996

Synergistic induction of anchorage-independent growth of NIH3T3 mouse fibroblasts by cysteine proteinase inhibitors and a tumor promoter.

We have previously reported that Ras protein is a potent cysteine proteinase inhibitor. In order to examine whether the cysteine proteinase-inhibitory activity of Ras is involved in carcinogenesis, the effects of the following probes were investigated. Cystatin alpha is a cysteine proteinase-specific inhibitor and has some amino acid sequence homology with Ras. Ras has a CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid) at the carboxyl terminus, which is indispensable for the biological activity. Thus, cystatin alpha carrying a CAAX motif (cystatin alpha-CVLS) was examined. A v-Ha-Ras deletion mutant, Ras delta 42-49, has undetectable GTP binding activity, yet it retains a similar protease inhibitory activity to that of wild-type v-Ras. These genes were inserted into a eukaryotic inducible expression vector and transfected into NIH3T3 cells. The expression was effectively induced by treatment with a glucocorticoid hormone, dexamethasone. The expression of cystatin alpha-CVLS or Ras delta 42-49 alone induced neither transformation nor morphological changes. However, when their expression was induced in the presence of a tumor-promoting phorbol ester, a remarkable increase in the anchorage-independent growth was observed in cystatin alpha-CVLS- and Ras delta 42-49-transfected clones. These results suggest that cysteine proteinase inhibitors and a tumor promoter synergistically transformed NIH3T3 cells. It is thus possible that the cysteine proteinase-inhibitory activity of Ras might play a key role in the early stage of carcinogenesis.

3T3 Cells↗
PubMed1996

Antitumor effect induced by the expression of granulocyte macrophage-colony stimulating factor gene in murine colon carcinoma cells.

Murine colon carcinoma cells which secrete several kinds of cytokine after retroviral transduction with corresponding genes, were examined for their antitumor effects in syngeneic mice. The mice inoculated with granulocyte macrophage-colony stimulating factor (GM-CSF) producer cells showed not only prolonged survival but also reduced tumorigenicity. The antitumor effect caused by the expression of interleukin-4 was less than that of GM-CSF, and interleukin-6 producer cells did not show any effects on the survival of the host animals. Histological examination of the GM-CSF-producing tumor revealed predominant infiltration of neutrophils and necrotic change of the tumor. The present study indicates the feasibility of cancer gene therapy with the expression of GM-CSF gene in tumor cells.

Animals↗
PubMed1996

Murine colon carcinoma cells engineered to produce human interleukin-2 induce tumor-specific anti-tumor response.

Murine colon carcinoma cells (colon 26) transduced by a retrovirus vector with the human interleukin-2 (IL-2) cDNA were studied for their tumorigenicity. Although cell growth in vitro was not affected by integration of the IL-2 gene, s.c. tumors of IL-2-producing colon 26 cells (H2) in syngeneic mice regressed spontaneously after producing small masses. Histological examination of the sites of tumor rejection revealed predominant infiltration of macrophages around the tumor necrotic mass. Subsequent challenge with parent colon 26 cells, but not with Meth A cells (fibrosarcoma of the same genetic background), did not result in tumor formation in mice which had been protected against H2 cells. Inoculation of H2 cells into syngeneic nude mice resulted in tumors with a retarded growth rate. Taken together, T cell-dependent, tumor-specific immunity is obtained by local IL-2 secretion around colon tumors, and this experimental animal model gives us a clue(s) for investigating host anti-tumor responses by cytokine production.

Animals↗
PubMed1996

Nuclear localization of procathepsin L/MEP in ras-transformed mouse fibroblasts.

It has been well-documented that secretion of procathepsin L is enhanced in ras-oncogene-transformed cells. In the present study, intracellular localization of cathepsin L was investigated by cell fractionation using Nonidet P-40 followed by immunoblot analysis. The results showed that a significant amount of procathepsin L was detectable in the nuclear fraction of Ha-ras, Ki-ras- and erbB2-transformed NIH3T3 mouse fibroblasts while procathepsin L was detected only in the cytoplasmic fraction of NIH3T3 cells and v-mos-transformed cells. These results suggest that the processing and translocation of cathepsin L are seriously impeded in ras- and erbB2-transformed cells.

3T3 Cells↗
PubMed1996

Difference in cytokine production in acute and chronic rejection of rat lung allografts.

In Brown Norway to Lewis rat lung transplantation, short-term administration of cyclosporine produces permanent adoption of allografts; however, the adopted grafts show symptoms of chronic rejection. To clarify the difference in cytokine production in acute an chronic rejection of the allografts, an immunohistochemical study was performed. In acute rejection, positive cells for respective cytokines were observed in infiltrating cells, increasing in number as the days after transplantation passed, and reaching a maximum on the fifth day. The strongest reactivity was observed perivenously. In chronic rejection, TNF-alpha positive cells were observed in the perivascular and peribronchial regions, especially around class II positive epithelia. The number of positive cells was, however, less than that in the vascular phase of acute rejection. Few cells were positive for IL-1 beta, IFN-gamma and, unexpectedly, for IL-4. These facts indicate the functional difference of infiltrating cells between acute and chronic rejection.

Animals↗
PubMed1996

Loss of Raf-1-binding activity of v-Ha-Ras by the deletion of amino acid residues 64-72 and 143-151.

In order to elucidate the molecular events in signal transduction, examination of the interaction between Ras and Raf-1 seems crucial. Many Raf-1 mutants have been investigated in terms of their binding activities to Ras, where only a few Ras mutants have been examined thus far. We have investigated the Raf-1-binding activities of v-Ha-Ras and 21 insertion/deletion mutants of this protein. The results show that the mutants have varying levels of Raf-1-binding activity that are related neither to their transforming activity nor to their guanine nucleotide-binding activity. Deletion in the effector domain of Ras did not completely abolish Raf-1-binding, whereas the deletion in amino acid residues 64-72 or 143-151 resulted in complete loss of Raf-1-binding activity.

Mutagenesis↗
PubMed1996

Interaction of rat Cdc37-related protein with retinoblastoma gene product.

By using in vitro binding assays and the yeast two-hybrid system, we have found that a full-length rat Cdc37-related protein (RCdc37) could associate specifically with the retinoblastoma susceptibility gene product (pRB). A series of GST-RCdc37 deletion mutants was constructed to define the amino acid sequence required for the interaction with pRB. A GST-RCdc37 (-20 approximately 229) possessed an activity to associate with pRB, whereas GST-RCdc37 (-20 approximately 165) lost its activity, indicating that the amino acid sequence between 166 and 229 of RCdc37 was essential for the association with pRB. Interestingly, there exists a highly conserved pRB-binding motif (LXCXE; X = any amino acid) that is essential for pRB binding of SV40 large T antigen, E1A, and E7 proteins. A similar experiment using a pRB deletion mutant revealed that the carboxy-terminal portion of pRB was required for binding to RCdc37.

Amino Acid Sequence↗
PubMed1996

Cloning of mouse DAN cDNA and its down-regulation in transformed cells.

Recently, we have demonstrated that DAN gene product exhibits a tumor-suppressive activity in vitro. We report here the cloning and sequencing of a mouse DAN cDNA that contains the entire coding region. Sequence analysis revealed that mouse DAM cDNA is 1691 nucleotides in length and contains an open reading frame of 178 amino acids. The deduced mouse DAN protein sequence shows 96% and 93% identity with the counterparts isolated from rat 3Y1 fibroblasts and normal human lung, respectively. Genomic Southern blot hybridization indicated that DAN gene exists as a single copy in the mouse genome. The expression of DAN gene was suppressed in a variety of transformed NIH3T3 cells when compared with that in the parental NIH3T3 cells.

3T3 Cells↗
PubMed1996

The clinical features of Tourette's disorder with obsessive-compulsive symptoms.

Twenty-three patients with Tourette's disorder (13 with obsessive-compulsive symptoms [OCS] and 10 without) were comparatively investigated. In contrast to OCS-free Tourette's disorder patients, those with OCS were found to be characterized by (i) a higher incidence of volatile temper, (ii) a higher incidence of compulsive tics, (iii) a higher incidence of perinatal disorders and brain wave abnormalities, (iv) a higher severity as rated using the Severity Scale, and (v) a higher prevalence of complications, especially of developmental disorders. Of the subjects with OCS-accompanied Tourette's disorder, approximately half had developed OCS by the onset of tics. These findings suggest the likelihood that OCS-accompanied Tourette's disorder is more strongly associated with organic cerebral disorders, independently of sites of tic disorders, than is OCS-free Tourette's disorder.

Adolescent↗
PubMed1995

Sequence-specific DNA binding activity of rat cdc37-related gene product.

The rat cdc37-related gene product (RCdc37), which is possibly involved in the regulation of cell cycle progression, contains a putative basic/leucine zipper (bZIP) domain in its N-terminal portion. In this study, we have identified a rat genomic sequence which can interact with RCdc37 using an in vitro binding assay. The specificity of this interaction was confirmed by gel retardation experiments. These results raise a possibility that RCdc37 might play an important role in the control of cell cycle progression via a sequence-specific DNA binding mechanism.

Amino Acid Sequence↗
PubMed1995

DAN gene product has an affinity for Ni2+.

Differential screening-selected gene aberrative in neuroblastoma (DAN) encodes a protein which possesses metal binding motifs. Glutathione S-transferase DAN fusion protein had an ability to bind to Ni(2+)-immobilized affinity resin. Truncation of the C-terminal region including a (HX)n repeat of DAN caused a loss of binding ability to the affinity resin, suggesting that this region is essential for Ni(2+)-binding. DAN produced in cultured rat cells also had an affinity for Ni2+. Cross-linking experiments demonstrated that the C-terminal region might function as a protein-protein interacting domain.

Amino Acid Sequence↗
PubMed1995

Purification and characterization of recombinant hamster tissue complement C1s.

Hamster complement C1s cDNA was inserted into expression plasmid BCMGSNeo, and transfected to SEA7 cells, A31 mouse fibroblasts transformed by polyoma virus. The transfectant secreted a large amount of recombinant C1s that was activated in the serum free culture medium and hydrolyzed acetyl-Gly-L-Lys-naphthyl ester (AGLNE). C1s was purified to a homogeneity from the culture medium of the transfectant by DEAE-Sephadex, Dymatrex orange A and size-exclusion HPLC. Purified hamster C1s consumed human complement in hemolytic assay and hydrolyzed gelatin in enzymography. To investigate the enzymic action of C1s at molecular levels, several antibodies were prepared against hamster C1s. One peptide (amino-acid residues 379-391) and two peptides (amino-acid residues 478-496 and 560-583) corresponding to the heavy and the light chain, respectively, were synthesized. The amino-acid sequences of these regions is not conserved between hamster and human C1s. Antibodies against these peptides were raised in rabbits. The anti-peptide antibodies bound specifically to hamster serum and recombinant C1s but not to human C1s. They inhibited the esterase activity of recombinant C1s to varying degrees depending on each antibody's binding site.

Amino Acid Sequence↗
PubMed1995

A region of consistent deletion in neuroblastoma maps within human chromosome 1p36.2-36.3.

Deletion of the short arm of human chromosome 1 is the most common cytogenetic abnormality observed in neuroblastoma. To characterize the region of consistent deletion, we performed loss of heterozygosity (LOH) studies on 122 neuroblastoma tumor samples with 30 distal chromosome 1p polymorphisms. LOH was detected in 32 of the 122 tumors (26%). A single region of LOH, marked distally by D1Z2 and proximally by D1S228, was detected in all tumors demonstrating loss. Also, cells from a patient with a constitutional deletion of 1p36, and from a neuroblastoma cell line with a small 1p36 deletion, were analyzed by fluorescence in situ hybridization. Cells from both sources had interstitial deletions of 1p36.2-36.3 which overlapped the consensus region of LOH defined by the tumors. Interstitial deletion in the constitutional case was confirmed by allelic loss studies using the panel of polymorphic markers. Four proposed candidate genes--DAN, ID3 (heir-1), CDC2L1 (p58), and TNFR2--were shown to lie outside of the consensus region of allelic loss, as defined by the above deletions. These results more precisely define the location of a neuroblastoma suppressor gene within 1p36.2-36.3, eliminating 33 centimorgans of proximal 1p36 from consideration. Furthermore, a consensus region of loss, which excludes the four leading candidate genes, was found in all tumors with 1p36 LOH.

Base Sequence↗
PubMed1995

Overexpression of DAN gene product in normal rat fibroblasts causes a retardation of the entry into the S phase.

Differential screening-selected gene aberrative in neuroblastoma (DAN) gene (previously named N03 gene), whose expression is significantly reduced in transformed cells, has recently been demonstrated to have a tumor-suppressive activity in vitro. In order to investigate biological roles of DAN gene product in normal rat fibroblasts (3Y1), marker-selected transfectants that expressed the high amount of DAN gene product were generated from 3Y1 cell lines. These clones did not exhibit morphological changes compared with parental 3Y1 cells; however, they showed a decrease in growth rate and a remarkable reduction in saturation density. Cell cycle analysis revealed that the overexpression of DAN gene product causes the retardation of the entry into the S phase. These results suggest that DAN gene product may have an important role in regulation of the entry of cells into the S phase.

Animals↗
PubMed1995

Increase of cyclin B by overexpression of cystatin alpha.

Degradation of cyclin B was effectively suppressed when cells were treated with ALLN (N-acetylleucylleucylnorleucinal) which inhibits proteasome, calpain and cysteine proteinase cathepsins. In order to examine which protease degrades cyclin B, the effect of a cathepsin inhibitor, cystatin alpha, was investigated. The cystatin alpha gene was inserted into an inducible expression vector, pMSG, and transfected into NIH3T3 mouse fibroblasts. The expression of cystatin alpha was induced effectively in the transfected cells after treatment with dexamethasone. Overexpression of cystatin alpha resulted in an increase of the amount of cyclin B, suggesting that cysteine proteinase cathepsins might be involved in the degradation of cyclin B.

3T3 Cells↗
PubMed1995

Suppression of syntheses of high molecular weight nonmuscle tropomyosins in macrophages.

In mouse fibroblasts, at least five TM isoforms are identified and they can be grouped into the high (TM1, TM2, and TM3) and low molecular weight TM isoforms (TM4 and TM5). Suppression of one of the high molecular weight tropomyosin (TM) isoforms in nonmuscle cells is implicated to be one of the causes for disorganization of actin microfilament bundles and subsequent changes in cell motility and cell shape. In this study, we studied the expression of tropomyosin isoforms in macrophages that exhibit high motility and ability to change cell shape. Two-dimensional gel electrophoresis followed by Western blot analysis using polyclonal anti-TM antiserum revealed that the high molecular weight TM isoforms were lacking in both resident and activated mouse peritoneal macrophages. Analyses of newly synthesized TM isoforms, Northern blot analyses using isoform-specific cDNA probes, and immunostaining with monoclonal anti-TM antibody that recognizes only the high molecular weight TM isoforms also demonstrated that the syntheses of the high molecular weight TM isoforms (TM1, TM2, and TM3) were completely suppressed, whereas the low molecular weight TM isoforms (TM4 and TM5) were expressed in macrophages. These results indicate that macrophages intrinsically lack the high molecular weight TM isoforms. In order to obtain information about cellular localization of the low molecular weight TM isoforms in macrophages, they were immunostained with polyclonal anti-TM antiserum that recognizes both the high and low molecular weight TM isoforms. The results showed that the low molecular weight TM isoforms were co-localized with F-actin in punctate and short fibrous structures. In addition, we performed in situ hybridization analysis to examine localizations of the TM mRNAs in fibroblasts and macrophages. The results showed that TM mRNAs were localized throughout the cytoplasm.

Actin Cytoskeleton↗
PubMed1995

Molecular cloning and cell cycle-dependent expression of a novel gene that is homologous to cdc37.

A cDNA clone, termed N17, was isolated from a rat fibroblast 3Y1 cDNA library by a differential screening technique. The expression of the N17 gene was significantly increased in a variety of rapidly growing cells, including v-src-, v-Ha-ras-, or v-mos-transformed 3Y1 cells, when compared with parental 3Y1 cells. The N17 gene is present as a single copy in rat genome and is evolutionarily conserved among higher eukaryotes. The predicted open reading frame (ORF) encodes a polypeptide of 379 amino acids that exhibits a significant similarity with those of the cell cycle control protein Cdc37. The amount of N17 mRNA starts to be increased in the late G1 phase and the same level was retained until just before the G2/M phase. Taken together, these results suggest that N17 gene product may play a crucial role in the cell cycle control of 3Y1 cells.

Amino Acid Sequence↗