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Biomedical subjects

Y Ikawa

Publications and source records attributed to Y Ikawa.

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

Transformation suppressor genes.

Flat revertants with reduced malignancy in vivo can be isolated from Kirsten sarcoma virus-transformed NIH3T3 cells following transfection with a normal human fibroblast cDNA expression library. We have recovered from one such revertant a 1.8 kb cDNA clone, "Krev-1", that possesses revertant-inducing activity in transfection assay. The Krev-1 cDNA has the capacity to encode a protein with a calculated molecular weight of 21,000 having strong structural similarity to ras proteins (ca. 50% homology), especially in their GTP/GDP-binding, effector-binding and membrane attachment domains. Point-mutations of Krev-1 protein at the 12th and the 59th amino acid positions from the aminoterminus potentiated the transformation suppressor activity by 2-5 times, and point-mutations within the putative effector-binding domain and at the C-terminal acylation site reduced the activity.

Amino Acid Sequence↗
PubMed1989

Further phenotypic characterization of target cells for bovine leukemia virus experimental infection in sheep.

To determine the phenotype of target cells for bovine leukemia virus (BLV) infection in sheep, we analyzed blood lymphocytes from BLV-infected clinically healthy and leukemic sheep by use of monoclonal antibodies. In clinically healthy and leukemic sheep that were BLV-infected, the blood concentration of T lymphocytes was within normal values, but the number of B lymphocytes was increased in several cases. In addition, the number of blood lymphocytes expressing the BLV antigen correlated well with that of B lymphocytes. Double immunofluorescence staining demonstrated that lymphocytes expressing BLV antigens bore B-cell but not T-cell surface markers. Moreover, neoplastic cells in the lymph nodes of leukemic sheep were stained immunohistochemically with an anti-B monoclonal antibody but not with any of anti-T monoclonal antibody tested, indicating that tumor cells are of B-lymphocyte origin. Collectively, these results show that BLV antigen-positive cells obtained from BLV-infected sheep that have no clinical signs and BLV-induced lymphosarcoma cells belong to the B-lymphocyte lineage.

Animals↗
PubMed1988

Possible involvement of two signaling pathways in induction of neuron-associated properties by v-Ha-ras gene in PC12 cells.

Harvey sarcoma virus induces a number of neuron-associated properties in a nerve growth factor-responsive cell line, PC12 (Noda, M., Ko, M., Ogura, A., Liu, D., Amano, T., Takano, T., and Ikawa, Y. (1985) Nature 318, 73-75). We investigated the mechanism of this phenomenon using PC12 sublines cotransfected with a plasmid containing v-Ha-ras gene under control of a hormone-responsive enhancer/promoter element of the mouse mammary tumor virus long terminal repeat (pM14-1) and a plasmid encoding G418 resistance (pSV2neo). Extent of the expression of neuron-associated properties in several cell clones after the addition of dexamethazone (DEX) seems to correlate well with the levels of the v-Ha-ras gene expression. After the induction of v-Ha-ras expression with DEX in these cell lines, sustained elevation of the levels of cAMP as well as of phosphatidylinositol (PtdIns) metabolites, inositol trisphosphate, and diacylglycerol, is observed. Physiological significance of this phenomenon is confirmed by the observation that dibutyryl cAMP and phorbol-12-myristate 13-acetate synergistically induces the expression of neuron-associated properties in PC12 cells. In control PC12 sublines transfected with pSV2neo alone, DEX shows no effects on their cell morphology and the levels of cAMP and the PtdIns metabolites, although these control cell lines are competent to the effects of dibutyryl cAMP and phorbol ester. The priming activity known to be associated with nerve growth factor is also observed with v-Ha-ras as well as with dibutyryl cAMP plus phorbol ester but not with dibutyryl cAMP or phorbol ester alone. The observations suggest that the role of v-Ha-ras gene product in this system may involved simultaneous activation of the two signaling pathways, those mediated by cAMP and by PtdIns turnover.

Acetylcholinesterase↗
PubMed1988

Characterization of erythropoietin receptor on erythropoietin-unresponsive mouse erythroleukemia cells.

A membrane receptor for erythropoietin was identified in various erythropoietin-unresponsive mouse erythroleukemia cells. Scatchard analyses of the binding of human 125I-labeled erythropoietin to T3C1-2-0, K-1, GM86 and 707 cells showed the presence of a single class of binding sites with apparent Kd values of 0.27-0.78 nM, which are slightly higher than those of erythropoietin-responsive cells. The number of binding sites varied from 110 to 930 per cell. Crosslinking of 125I-erythropoietin to its binding sites with disuccinimidyl suberate revealed the existence of a single binding protein with molecular mass of 63 kDa. No binding sites with higher molecular mass, as observed in erythropoietin-responsive cells, were detected, nor was any specific binding observed to the non-erythroid hematopoietic cell or to the human erythroleukemia cells examined.

Animals↗
PubMed1988

Active ras and myc oncogenes can be compatible, but Sv40 large T antigen is specifically suppressed with normal differentiation of mouse embryonic stem cells.

The pathobiological effects of oncogenes on normal differentiation of mouse embryonic stem cells from 4-day embryos were examined by introducing active ras, myc, and SV40 large T genes, all driven by mouse metallothionein I enhancer and promoter. Stem cell clones R5, M3, and T2 for ras, myc, and SV40 T genes, respectively, were particularly chosen for analyses because of their higher levels of transgene expression and their diploid chromosomal constitutions. These stem cells were then introduced into host 4-day embryos and the embryos were allowed to develop in the uterus of foster mothers. The stem cells colonized the tissues as extensively as the parent cells and gave rise to adult chimera with no apparent loss or abnormality of the embryos. The active ras and myc oncogenes introduced were expressed not only in the stem cells, but also in the developing embryos and in a variety of tissues of adult chimeras. However, although T antigen was originally expressed in the stem cells, it was not expressed in either developing embryos or tissues of adult chimeras. Induced by retinoic acid treatment in vitro or by subcutaneous grafting, this suppression of T-gene expression was also confirmed in differentiated progeny cells from several stem cell clones expressing T antigen. Permanent lines of fibroblast-like cells could be established at higher frequency from primary cultures of tissues of chimera, subcutaneous differentiated cells, and in vitro differentiated cells derived from T2 cells, and all these clones reexpressed T antigen. The results suggest that active myc and ras genes can be compatible with normal differentiation of the stem cells, but the expression of T antigen is specifically suppressed with recognition of its coding domain.

Animals↗
PubMed1988

Identification of putative endogenous proviral templates for progenitor mink cell focus-forming (MCF) MuLV-related RNAs.

Murine leukemia virus (MuLV)-related RNAs exhibiting different env deletions are believed to participate in the generation of leukemogenic mink cell focus-forming (MCF) viruses. We have cloned an endogenous MuLV provirus from AKR/J mouse DNA, designated as A-2, which may serve as template for the env-deleted E2 MuLV RNA, expressed in GIX+ mice (D.E. Levy et al., J. Virol. 56, 691-700 (1985]. We have also isolated an endogenous MCF-related DNA, A-1, which shared close sequence homology with the 7.2-kb RNA expressed in AKR mice (F. Laigret et al., J. Virol. 62, 376-386 (1988] and sustained an identical env deletion. The data indicate that putative precursor MCF-related RNAs are transcribed from a heterogenous family of env-deleted endogenous MuLV DNAs.

Base Sequence↗
PubMed1988

Immunogenicity of a recombinant vaccinia virus expressing envelope a glycoprotein of bovine leukaemia virus.

We constructed a recombinant vaccinia virus (RVV) expressing envelope (env) glycoprotein (gp51) of bovine leukaemia virus (BLV): the expression of gp51 was detected by Western blot of the lysates of rabbit kidney cells infected with the RVV. The rabbits inoculated intradermally with the RVV alone failed to induce detectable anti-gp51 antibodies even 10 weeks after immunization. However, when these animals were boosted with inactivated BLV virion in saline, significant levels of anti-gp51 antibodies were induced as shown both in Western blot and immunodiffusion analyses. In these animals, antibodies against gag product (p24) were not detected. On the other hand, the rabbits inoculated with wild-type vaccinia virus and boosted similarly three times with the BLV virion in saline did not induce detectable anti-gp51 antibodies at all. The present experiment revealed that the RVV possessed the capability to endow immunological memory without inducing apparent anti-gp51 antibody responses, meaning that the RVV activated helper T cells far more strongly than B cells. The applicability of the RVV to vaccine development is discussed.

Animals↗
PubMed1988

Down-regulation of c-myb gene expression is a prerequisite for erythropoietin-induced erythroid differentiation.

The role of nuclear protooncogenes during erythroid cell differentiation was examined by transfecting exogenous c-fos and c-myb genes into mouse erythroleukemia cells, which can be induced to differentiate either with erythropoietin (Epo) or dimethyl sulfoxide. Expression of exogenous c-myb or c-fos oncogene completely inhibited Epo-induced erythroid differentiation but only partially inhibited dimethyl sulfoxide-induced differentiation. Normally Epo-induced differentiation leads to a drastic decline of c-myb mRNA levels and an increase of c-myc transcripts in the early stage of differentiation. Cells expressing exogenous c-fos gene, however, maintained high levels of c-myb mRNA after Epo treatment. This high level of c-myb transcripts was found to be due to block of transcription shutoff (or transcriptional activation) rather than to mRNA stabilization. It is concluded that the down-regulation of endogenous c-myb gene expression is a prerequisite for commitment of Epo-induced erythroid differentiation and that expression of c-myb gene may be indirectly regulated by c-fos gene product. We also concluded that early down-regulation of c-myc gene expression is not essential for erythroid differentiation and that gene regulation of chemically induced erythroid differentiation may differ from that of Epo-induced differentiation.

Animals↗
PubMed1987

Driven by the same Ig enhancer and SV40 T promoter ras induced lung adenomatous tumors, myc induced pre-B cell lymphomas and SV40 large T gene a variety of tumors in transgenic mice.

Different types of tumors developed in transgenic mice following the introduction of the entire coding region of ras, myc or SV40 large T gene (T) linked to the same regulatory unit, consisting of a human immunoglobulin gene enhancer (Ig) and SV40 early gene promoter (Tp) with a 21-bp repeat. All the 12 transgenic mice harboring the intact T gene developed a variety of tumors including choroid plexus tumor, B cell lymphoma, histiocytic lymphoma, thymoma and others. This suggests that the Ig/Tp regulatory unit has transcriptional activity in these heterologous tissues. With this regulatory unit, myc gene induced solely pre-B cell lymphomas (five out of nine mice). Contrary to our expectation, however, the mutated ras gene induced lung adenomatous tumors in six out of eight transgenic mice over the 10-month observation period; the tumors are histologically comparable to adenocarcinomas in man. The tumors developed as early as 4 weeks after birth and the introduced ras gene was as efficiently expressed in both normal and neoplastic bronchioloalveolar epithelial cells as in normal lymphoid cells. An unidentified secondary event thus appears to be necessary for these ras-expressing cells to become neoplastic, as observed for myc (Leder et al., 1986). In a variety of tumors induced by Ig/Tp-T, on the other hand, T gene was expressed only in the tumor cells, but not in normal cells. Thus, derepression of T gene in normal cells appears to be closely related to their malignant change as observed in development of pancreatic acinar cell tumors by the T gene (Ornitz et al., 1985). These results suggest that ras and myc oncogenes penetrate differentially specific types of cells, while the SV40 T gene is tumorigenic in a variety of cell types.

Adenoma↗
PubMed1987

Production of recombinant rat viruses as a method of oncogene isolation in coculture medium.

A simple oncogene isolation was proved using the SD1-T rat embryonic cell line. The SD1-T cell line, which releases endogenous rat leukemia virus, was cocultured with (a) normal rat kidney cells transformed by cloned v-mos DNA, (b) the rat mammary tumor cell line (63SP), or (c) normal rat kidney cells transformed by 63SP DNA. Within 1 mo, oncogenic viruses were recovered from all three coculture supernatants. During this period, increase of oncogenic transcripts was observed in the cocultured cells. The oncogenic viruses appeared to contain the mos gene in cocultures (a) and ras-related sequences in cocultures (b) and (c). The emergence of virus containing mos from mos DNA-mediated normal rat kidney transformants demonstrated "rescue" of the active cellular oncogene by the rat leukemia virus. This coculture system seems to facilitate "rescue" of oncogenes functioning in the tumor and transformed cells.

Animals↗
PubMed1987

Mouse embryonic stem cells exhibit indefinite proliferative potential.

The proliferative potential of embryonic stem cells was examined. In contrast to the current concept of the finite life-span being the hallmark of normal cells, we have been able to maintain these embryonic stem cells in vitro up to about 250 cumulative doublings with no indication of "crisis" or transformation. These cells could be considered normal on the basis of: (1) their apparently normal diploid karyotype, (2) their ability to extensively colonize embryos without causing tumors and developmental anomalies, and (3) their ability to form normal gametes when differentiated into the germ-line. These results suggest that embryonic stem cells prior to differentiation into germ and somatic cells are indeed immortal.

Animals↗
PubMed1987

Specific binding of erythropoietin to its receptor on responsive mouse erythroleukemia cells.

Erythropoietin (Epo) is a glycoprotein factor that specifically regulates the proliferation and differentiation of erythroid progenitor cells. Here we describe the isolation of Epo-responsive mouse erythroleukemia cell line SKT6, the characterization of the specific binding of biologically active 125I-labeled human Epo (125I-Epo) to its membrane receptor, and, finally, report information concerning the molecular structure of the receptor. About 75% of erythroid colony-forming precursor cell-like colonies derived from SKT6 cells were hemoglobin-positive after 3- to 4-day exposure to Epo in methylcellulose culture. Radioiodinated Epo bound specifically to SKT6 cells, and Scatchard analysis of the data showed a high affinity for 125I-Epo (Kd = 0.15 nM) but displayed only a small number of specific receptors (approximately equal to 470 per cell). Membrane components that specifically interact with 125I-Epo were identified by covalent crosslinking with disuccinimidyl suberate, and three receptor species with apparent Mr 63,000, 94,000, and 119,000 were found in membrane from SKT6 cells, suggesting the complex structure of the receptor molecules. Specific bindings were also detected in all of the Epo-unresponsive Friend erythroleukemia cells examined, and cross-linking study revealed the presence of only the 63,000 species as a binding site.

Animals↗
PubMed1987

Structure of a defective provirus of bovine leukemia virus.

Defective proviruses of bovine leukemia virus (BLV) in the genomes of infected cells were investigated by using Southern blotting hybridization analysis with various portions of a cloned BLV DNA as probes. When nine independent tumors of enzootic bovine leukosis with a single proviral copy per cell were examined, a single defective provirus of BLV was found in one tumor and also in a bovine B cell line derived from this tumor. Hybridization analysis of this defective provirus revealed that it underwent deletion between the pol and env genes and contained no major deletion in the other regions.

Animals↗
PubMed1987

The bovine leukemia virus X region encodes a trans-activator of its long terminal repeat.

We constructed a fusion plasmid, pMX-I, by which the major open reading frame, X-I, of the bovine leukemia virus (BLV) X gene was expressed under control of the mouse metallothionein promoter. pMX-I was cotransfected into CV1 monkey kidney cells together with another construct containing the BLV long terminal repeat (LTR) linked to the chloramphenicol acetyltransferase (CAT) structural gene. The result of assay of CAT synthesis suggests that the X-I product functions as a trans-acting activation factor of the BLV LTR.

Acetyltransferases↗
PubMed1986

Decrease in glucose transport activity of Friend erythroleukemia cell caused by dimethylsulfoxide, a differentiation-inducing reagent.

A transport system for D-glucose was found in a Friend erythroleukemia cell line, T-3-C1-2-O and was characterized as a facilitated diffusion system. D-Glucose transport activity showed a half-saturation concentration of 2.2 mM and was inhibited by mercuric ions, cytochalasin B, phloretin, and stilbestrol, but was not strongly inhibited by phloridzin. Transport of 3-O-methyl-D-glucose was faster than D-glucose and the intracellular concentration of the sugar was found to reach the concentration in the assay medium. The treatment of cells with a differentiation-inducing reagent, dimethylsulfoxide(Me2SO), for 24 h caused a marked decrease in glucose transport activity due to a decrease in Vmax. In an induction-insensitive Friend cell line, T-3-K-1, D-glucose transport activity was low in untreated cells and Me2SO treatment did not cause a significant decrease in transport activity. The results obtained in this study indicate that the decrease in glucose transport activity is not due to the direct effect of Me2SO on transport activity, but is associated with the induction of differentiation. By immunoblotting cell lysates of T-3-C1-2-O cells using antibody to human erythrocyte glucose transporter, a single major band having a molecular weight of 52,000 was detected, which may be a glucose transporter in Friend cells.

Animals↗
PubMed1986

Protease gene structure and env gene variability of the AIDS virus.

The protease gene structure and the env gene variability have been precisely compared between the AIDS virus and members of the HTLV/BLV family. The conserved amino acid sequence (LVDT) which is repeated in the proteases of the HTLV/BLV family is not repeated in AIDS virus. Comparative analysis of the env gene sequences reveals the striking fact that the env gene of AIDS virus is 8-12-times more variable than those of the HTLV/BLV family. Within the AIDS virus env gene, the surface glycoprotein region is more liable to vary than is the transmembrane region; unexpectedly, however, this liability is not a characteristic feature of the AIDS virus because it is more prominent in other retroviruses including members of the HTLV/BLV family.

Acquired Immunodeficiency Syndrome↗