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Biomedical subjects

K Ozato

Publications and source records attributed to K Ozato.

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

Regulation of mouse CYP1A1 gene expression by dioxin: requirement of two cis-acting elements during induction.

The mouse cytochrome P1450 (CYP1A1) gene is responsible for the metabolism of numerous carcinogens and toxic chemicals. Induction by the environmental contaminant tetrachlorodibenzo-p-dioxin (TCDD) requires a functional aromatic hydrocarbon (Ah) receptor. We examined the 5'-flanking region of the CYP1A1 gene in mouse hepatoma Hepa-1 wild-type cells and a mutant line having a defect in chromatin binding of the TCDD-receptor complex. We identified two cis-acting elements (distal, -1071 to -901 region; proximal, -245 to -50 region) required for constitutive and TCDD-inducible CYP1A1 gene expression. Three classes of DNA-protein complexes binding to the distal element were identified: class I, found only in the presence of TCDD and a functional Ah receptor, that was heat labile and not competed against by simian virus 40 (SV40) early promoter DNA; class II, consisting of at least three constitutive complexes that were heat stable and bound to SV40 DNA; and class III, composed of at least three constitutive complexes that were thermolabile and were not competed against by SV40 DNA. Essential contacts for these proteins were centered at -993 to -990 for the class I complex, -987, -986, or both for the class II complexes, and -938 to -927 for the class III complexes. The proximal element was absolutely essential for both constitutive and TCDD-inducible CYP1A1 gene expression, and at least two constitutive complexes bound to this region. These data are consistent with the proximal element that binds proteins being necessary but not sufficient for inducible gene expression; interaction of these proteins with those at the distal element was found to be required for full CYP1A1 induction by TCDD.

Animals↗
PubMed1988

Establishment of cell lines from somite stage mouse embryos and expression of major histocompatibility class I genes in these cells.

To study the regulation of MHC class I gene expression during embryonic development, we have characterized a number of clonal cell lines derived from somite stage mouse embryos that were established with or without infection by several transforming retroviruses in combination with murine leukemia viruses. Unlike embryonal carcinoma (EC) cells that have been used as a model for early embryos, the cell lines derived from somite stage embryos are negative for stage specific embryonic Ag-1 and do not appear to differentiate after retinoic acid treatment. Morphology varies from clone to clone and is distinct from that of F9 and other EC cells. In agreement with previous findings in in vivo embryos, expression of surface MHC class I antigen in 57 new clones is either undetectable or low (with variability). All of the clones respond to the addition of interferons and express MHC class I antigens at high levels, but the kinetics of mRNA accumulation vary considerably. To examine the basis of the generally low or absent MHC class I gene expression in these cells, we tested promoter activity of a MHC class I gene by CAT assay after transient DNA transfection. Regardless of the basal levels of mRNA or surface Ag, CAT activity directed by various portions of the 5' flanking region of the MHC class I gene was uniformly low. The cells showed neither the negative nor the positive regulation of MHC class I genes that had been noted respectively for EC cells and for cells expressing the Ag constitutively. The pattern seen in the new cell lines suggests that there is an intermediate stage in the developmental regulation of MHC class I gene expression that may operate during the middle to late stage of fetal development.

Animals↗
PubMed1988

c-fos antisense RNA blocks expression of c-fos gene in F9 embryonal carcinoma cells.

To study the function of proto-oncogene c-fos, we prepared an antisense plasmid that expresses in mammalian cells c-fos antisense RNA which is complementary to the endogenous c-fos mRNA. Upon transfection into undifferentiated F9 EC cells, the antisense plasmid directed constitutive expression of a large amount of c-fos antisense RNA. These cells were very low in the basal level of c-fos message and were unable to induce c-fos message when stimulated with interferon or phorbol ester. The failure to induce c-fos message led to the blockade of c-fos protein expression in these cells. Thus, these cells represented a c-fos defective phenotype. The blockade of c-fos gene expression seen in antisense-cells could be caused by rapid degradation of the c-fos message, since c-fos mRNA expression was rescued in these cells when treated with protein synthesis inhibitor, cycloheximide. We found that expression of c-myc gene was down-regulated in c-fos antisense-cells: Although control undifferentiated F9 cells constitutively expressed a high level of c-myc message, the antisense cells had a much lower amount of c-myc mRNA. Since p53 and heat shock gene 70 were expressed at comparable levels in control and antisense cells, c-myc gene expression appears to be regulated by c-fos gene in F9 EC cells. Lastly, these antisense cells grew as rapidly as control F9 cells and underwent differentiation after retinoic acid treatment, indicating that c-fos expression is not a prerequisite for differentiation of F9 cells.

Animals↗
PubMed1988

Interferon-induced transcription of a major histocompatibility class I gene accompanies binding of inducible nuclear factors to the interferon consensus sequence.

Interferon (IFN) induces transcription of major histocompatibility class I genes by way of the conserved cis-acting regulatory element, termed the IFN consensus sequence (ICS). Binding of nuclear factors to the ICS was studied in gel mobility shift assays with the 5' upstream region of the murine H-2Ld gene. We found that the ICS binds a constitutive nuclear factor present in lymphocytes and fibroblasts regardless of IFN treatment. Within 1 hr after IFN treatment, new ICS binding activity was induced, which consisted of at least two binding activities distinguished by their requirement for de novo protein synthesis. Methylation interference and competition experiments showed that both constitutive and induced factors bind to the same approximately equal to 10-base-pair binding site within the ICS. Site-directed mutagenesis of H-2Ld-chloramphenicol acetyltransferase fusion genes showed that mutations in the binding site, but not in other regions of the ICS, abolish transcriptional activation of class I genes by IFN, providing evidence that specific binding of nuclear factors to the ICS is an essential requirement for transcriptional induction. Finally, we show that IFN-inducible genes of various species share a sequence motif that is capable of competing for the nuclear factors identified here. We propose that specific protein binding to the conserved motif represents a basic mechanism of IFN-mediated transcriptional induction of a number of genes.

Amino Acid Sequence↗
PubMed1988

Induction of c-fos gene expression by interferons.

We show that murine interferons (IFNs)-alpha/beta and -gamma induce expression of the protooncogene c-fos in F9 embryonal carcinoma cells, NIH-3T3 cells, and other tissue culture cells. The induction of c-fos mRNA is rapid and transient in that the mRNA appears within 15 min and disappears by 2 h following IFN treatment. The protein synthesis inhibitor cycloheximide (CHX) also stimulates rapid but more stable expression of c-fos mRNA in these cells. Treatment of these cells with a combination of CHX and IFNs results in superinduction of c-fos mRNA. In contrast, IFNs do not affect c-myc mRNA expression in these cells. These results suggest that c-fos gene expression plays a role in signal transduction following binding of IFNs to the receptors.

Animals↗
PubMed1988

Constitutive expression of c-fos antisense RNA blocks c-fos gene induction by interferon and by phorbol ester and reduces c-myc expression in F9 embryonal carcinoma cells.

To address the role of c-fos proto-oncogene we constructed a plasmid that allows constitutive expression of RNA complementary to c-fos mRNA, and stably introduced this plasmid into F9 embryonal carcinoma cells. Some F9 clones expressing c-fos antisense RNA had a reduced basal level of c-fos mRNA, and were unable to induce a c-fos mRNA as well as its protein when stimulated with phorbol ester or with interferon (IFN). Nevertheless, the ability to induce major histocompatibility class I genes following IFN treatment was not impaired in these clones. Clones expressing c-fos antisense RNA grew as rapidly as control F9 cells, and underwent differentiation after retinoic acid treatment. Unexpectedly, constitutive expression of c-myc mRNA was reduced on average by 10-fold in clones expressing c-fos antisense RNA. However, expression of the p53 gene and heat shock gene hsp 70 was not affected in these clones, indicating the existence of a specific regulatory linkage between c-fos and c-myc genes. Cycloheximide treatment led to induction of a large amount of c-fos mRNA in clones expressing c-fos antisense RNA as well as in control F9 clones. The amount of c-fos antisense RNA was also increased by cycloheximide treatment. We postulate that c-fos antisense RNA blocks expression of the endogenous c-fos gene by accelerating the degradation of c-fos mRNA and that cycloheximide treatment interferes with this degradation.

Cell Differentiation↗
PubMed1987

Introduction of H-2Dd determinants into the H-2Ld antigen by site-directed mutagenesis.

We used site-directed mutagenesis to localize serologically defined (s) and CTL (c)-defined alloantigenic determinants to discrete amino acid sequences of a murine MHC class I antigen. Based on the prediction that amino acid position 63-73 of the H-2Dd antigen forms s-allodeterminants, the H-2Ld gene was mutated in a sequential fashion to replace codons for amino acid positions 63, 65, 66, 70, and 73 with those of the H-2Dd amino acids. Epitopes of the mutant antigens expressed in L-cells were examined by the binding of a series of mAbs specific for the H-2Dd antigen. The mutant antigen M66 had substitutions at residues 63, 65, and 66, and resulted in the acquisition of a number of H-2Dd-specific s-epitopes. Mutant M70 had an additional substitution at residue 70, which led to the gain of multiple additional H-2Dd s-epitopes. Together, more than half of all the relevant H-2Dd s-epitopes were mapped into amino acid position 63-70 of the H-2Dd molecule, which was expressed in the mutant H-2Ld gene. The final mutation at residue 73 (M73) caused no new epitope gains, rather, a few Dd s-epitopes acquired by the preceding mutations were lost. All of the H-2Ld-specific s-determinants were retained in the mutant molecules, as were H-2Dd s-determinants specific for the alpha-2 or alpha-3 domains. Changes of these residues affected c-determinants defined by CTL. Anti-H-2Dd CTL cultures and an anti-H-2Dd CTL clone recognized the mutant H-2Ld molecules, M66 and M70. Some CTL clones generated against the Q10d molecule, which has an identical sequence to H-2Dd between residues 61 and 73, failed to recognize native H-2Dd or Ld but did crossreact with mutant Ld. While bulk-cultured anti-H-2Ld CTL cultures reacted strongly against M73, bulk-cultured H-2Ld restricted anti-vesicular stomatitis virus CTL did not. Finally, at the clonal level two of three anti-H-2Ld CTL clones lost reactivity with some or all of these mutant molecules. From these results we conclude that a stretch of amino acids from position 63 to 70 of the alpha-1 domain controls major s- and c-antigenic sites on the H-2Dd antigen and c-sites on H-2Ld antigen.

Amino Acid Sequence↗
PubMed1987

Induction of c-ets and c-fos gene expression upon antigenic stimulation of a T cell hybridoma with inducible cytolytic capacity.

Expression of cellular oncogenes was studied in a T cell hybridoma that undergoes cytolytic activation when stimulated by specific antigen or by anti-Thy-1 antibody. The activation occurs without induction of hybridoma proliferation, providing a model to examine oncogene expression during functional differentiation of lymphocytes. We found that c-fos and c-ets-1 mRNAs were transiently induced at high levels in the hybridoma 30 min and 4 h after stimulation, respectively. c-myc and c-ets-2 oncogenes were constitutively expressed in the hybridoma and their mRNA levels were unaffected during 4 h of stimulation, although c-myc expression was reduced in the later stage of stimulation. Inhibitors of T cell activation, cyclosporin A and anti-LFA-1 antibody, blocked the induction of c-fos and c-ets-1 mRNAs without reducing the levels of c-myc and c-ets-2. The results indicate that the functional activation of the CTL hybridoma is associated with induction of c-fos and c-ets-1 genes.

Animals↗
PubMed1987

Fine mapping of epitopes by intradomain Kd/Dd recombinants.

11 intradomain recombinants between H-2Kd and H-2Dd were produced using an original technique based on in vivo recombination in Escherichia coli. After transfection into mouse L cells, all these recombinants were expressed at high levels on the cell surface. The specificities of 77 mAbs were examined on these cell lines. mAbs could be organized in 12 groups. In each group, a small number of amino acids participating in the recognized epitope(s) were identified. In a few instances, noncontinuous epitopes comprising amino acids belonging to different domains of the antigen were found. The data thus obtained are compatible with those produced in previous exon-shuffling experiments, but permit a much more precise definition of recognized epitope(s).

Animals↗
PubMed1987

Transfer of antibodies directed to paternal major histocompatibility class I antigens from pregnant mice to the developing fetus.

The placental transmission of antibodies directed toward paternal MHC Class I antigens to the developing fetus was studied to assess their effect on the expression of MHC antigens during fetal development and on the development of immune function. 125I-monoclonal anti-paternal MHC antibodies injected i.v. into pregnant mice on day 15 of gestation were efficiently transferred to the fetus within 24 hr in a dose-dependent manner. Biochemical studies on the transferred radioactivity showed that intact antibodies accumulated in the fetus for up to 3 days after antibody injection. During the same period, antibodies were eliminated from the maternal system. The transfer and accumulation of anti-MHC antibodies were independent of the MHC haplotype of the fetus. The pathway of antibody transfer and the localization of transmitted antibodies in the fetus were studied by autoradiographic analysis of the entire fetoplacental unit 24 hr after the injection of anti-paternal MHC antibodies. Our results indicate that antibodies are transferred by way of the placenta and yolk sac, and reach the fetus predominantly via the circulation. Within the embryo proper, the highest levels of antibody were found in the order of blood greater than thymus greater than fetal liver. Most other fetal organs, with the exception of brain and cartilage, showed antibody accumulation, but to a lesser extent. This pattern of antibody distribution over different tissues was similar for allogeneic and syngeneic fetuses. These findings demonstrate that various fetal tissues, including developing lymphoid cells can be directly exposed to the maternally transmitted anti-MHC antibodies, with possible functional consequences on the development of the fetal immune system.

Animals↗
PubMed1987

New family of exon-shuffled recombinant genes reveals extensive interdomain interactions in class I histocompatibility antigens and identifies residues involved.

The specificities of an extensive panel of anti-H-2Dd monoclonal antibodies, which had been previously characterized using exon-shuffled H-2Dd/H-2Ld molecules and a number of anti-H-2DP antibodies, were examined using H-2Dd/H-2DP recombinants. The use of this new family of recombinant antigens revealed extensive interaction between the membrane-distal (N and Cl) domains of class I molecules. 20 out of 48 mAbs recognize complex epitopes formed by the interaction of these two domains. These antibodies exhibit a number of distinct patterns of crossreactivity with other class I proteins, revealing the presence of multiple epitopes within the region of domain interaction. Comparison of the data presented here with those from previous work allowed the identification of a small number of residues in the Cl domain that participate in the generation of complex epitopes involving both the N and Cl domains. The results are discussed in terms of the structural information available for these two domains.

Amino Acid Sequence↗
PubMed1987

Induction of major histocompatibility class I antigens by interferons in undifferentiated F9 cells.

Mouse embryonal carcinoma F9 cells, upon treatment with interferons (IFNs), express major histocompatibility (MHC) Class I antigens, which are otherwise not expressed in these cells. Both IFN-gamma and IFN-alpha/beta increase the steady-state level of Class I mRNA within 60 min of the treatment which leads to the subsequent surface expression of the H-2Kb and H-2Db antigens, suggesting that undifferentiated F9 cells express IFN receptors. IFNs induce Class I antigen expression in F9 cells in a highly selective manner: unlike retinoic acid treatment which also stimulates the antigen expression, IFNs induce neither morphological differentiation, increased binding of epidermal growth factor, nor reduction of expression of stage specific embryonic antigen. The effect of IFNs is reversible; removal of IFNs, even after prolonged exposures, results in a rapid loss of the Class I gene expression. Further, Class I mRNA induction is not inhibited by cycloheximide, suggesting possible independence from de novo protein synthesis. This Class I antigen induction in F9 cells is reminiscent of that observed in somite stage mouse embryos by IFN treatment and may offer a model system to study activation of MHC genes during development.

Animals↗
PubMed1987

Induction of xanthophores from non-pigmented dermal cells of xanthic goldfish in vitro.

To identify precursor cells of xanthophores (xanthoblasts), non-pigmented cells without any phenotypic traits as pigment cells were isolated from the dermal tissue of xanthic goldfish with an adult color pattern and cultured in a medium containing 1 mM db-cAMP or 0.25 U/ml ACTH and 10% carp serum. These non-pigmented cells differentiated into xanthophores which showed a dendritic morphology and contained a large quantity of fluorescent pteridines and numerous vesicular inclusions. Sepiapterin was the major component, and the vesicles contained fuzzy material in addition to small membranous elements. The fluorescent pattern and the morphological characteristics indicated that the differentiated pigment cells were xanthophores of larval type.

Adrenocorticotropic Hormone↗
PubMed1987

Binding of multiple nuclear factors to the 5' upstream regulatory element of the murine major histocompatibility class I gene.

Transcription of mouse major histocompatibility complex class I genes is controlled by the conserved class I regulatory element (CRE) in the 5' flanking region. The CRE, approximately 40 base pairs long, acts as a negative control element in undifferentiated F9 embryonal carcinoma cells which do not express the major histocompatibility complex genes. The same element, however, acts as a positive control element in cells expressing the genes at high levels. To investigate the molecular basis of the regulatory role of the CRE, we studied the binding of nuclear proteins to the CRE of the H-2Ld gene by gel mobility shift and methylation interference experiments. Nuclear extracts from L fibroblasts and LH8 T lymphocytes revealed three distinct factors that bind discrete sequences within the CRE. The three sequences correspond to the inverted and direct repeats within the CRE. In contrast, F9 extracts exhibited factor binding to only two of the three sequences and lack a major factor detected in the above two cell types. Protein-binding sites within each of the three sequences were identified by methylation interference experiments. These data were in full agreement with results obtained by a competition assay performed with a series of mutant oligonucleotides containing a few nucleotide substitutions in each of the three regions. The results illustrate complex DNA-protein interactions in which several independent proteins bind to overlapping sequences in the CRE in a cell type-specific fashion.

Animals↗
PubMed1987

Interferons increase transcription of a major histocompatibility class I gene via a 5' interferon consensus sequence.

Interferons (IFNs) augment expression of major histocompatibility class I genes in many cells. To study the effect of IFNs on transcription of class I genes, we prepared and tested activity of chloramphenicol acetyltransferase (CAT) hybrid genes in which the cat gene is under the control of the 5' flanking region of the murine H-2Ld gene. NIH 3T3 cells transiently transfected with a cat construct having the sequence from position -210 to -134 showed a four- to fivefold increase in CAT activity when treated with IFN-alpha/beta. This sequence contains the IFN consensus sequence (ICS) shared among IFN-inducible genes, as well as the class I regulatory element (CRE) that controls up and down regulation of class I gene expression. To determine the precise sequence requirement for the IFN action, the ICS and CRE were independently placed upstream of the class I or a heterologous simian virus 40 promoter, and CAT activity was tested. The ICS, but not the CRE, enhanced activity of both promoters by about twofold upon exposure to IFN-alpha/beta, although greater responses were noted when the ICS and CRE were combined. These results demonstrate that the ICS alone is capable of enhancing promoter activity in response to IFN-alpha/beta treatment and that the CRE exerts a synergistic effect. Further, we show that the ICS functions as an inducible enhancer since it acts regardless of its orientation and distance in the simian virus 40 promoter.

Acetyltransferases↗
PubMed1987

A burst of c-fos gene expression in the mouse occurs at birth.

Expression of the c-fos gene during murine perinatal development was studied. Before birth, all eight of the prenatal organs tested expressed undetectable or low levels of c-fos mRNA. On the day of birth, there occurred a 10- to 100-fold increase in the level of c-fos message in all of these organs. The expression was transient, in that 1 day after birth, the level of c-fos mRNA precipitously dropped. The c-fos gene expression at birth is unrelated to the expression of the c-myc gene and major histocompatibility complex class I genes, which display distinct kinetics during the perinatal development. The c-fos gene was also expressed locally and transiently in the gravid uterus 1 to 2 days prior to delivery. These results indicate that an event associated with birth induced c-fos gene expression in the mother and newborn.

Animals↗
PubMed1987

Fish plasminogen activators: their identification and characterization.

Immunoblots of proteins extracted from the skin of a small viviparous fish (Xiphophorus) showed that a monoclonal antibody against human urokinase recognizes multiple molecular weight species of antigens. The immunoaffinity-purified antigens had serine-protease activity for the hydrolysis of a chromogenic substrate and could convert human plasminogen to plasmin in a manner similar to that for human urokinase in vitro. Two antigens with apparent molecular weights of 55 and 50 kilodaltons that had been purified on a fibrin-Celite column were separable on SDS-polyacrylamide gels and were characterized as major plasminogen activators on fibrin-agar indicator plates. The 125I-tryptic peptide maps of both antigens were similar to that of human urokinase; therefore, the fish activators and human urokinase are structurally and functionally related.

Animals↗