PubMed Health⌕ Search

Biomedical subjects

H Iba

Publications and source records attributed to H Iba.

At least 19 recordsLinked to original sources
PubMed1991

Difference in transcriptional regulatory function between c-Fos and Fra-2.

Fra-2, one of the Fos-related antigens, is promptly expressed after the growth stimulation of fibroblasts, but its induction peak is later than that of c-Fos. In this report, we examined biochemical properties of Fra-2 and compared them with those of two other Fos family proteins, c-Fos and Fra-1. Like c-Fos and Fra-1, Fra-2 formed stable heterodimers with c-Jun, JunB or JunD in vitro and all these complexes had specific DNA-binding activity to AP-1-binding sites (AP-1 sites) or related sequences. When transiently introduced into a mouse embryonic carcinoma cell line, F9, with reporter genes containing the AP-1 site from the collagenase gene, fra-2 plus c-jun suppressed the transactivation by c-jun alone. This property of Fra-2 is in clear contrast to that of c-Fos, which stimulates the transcriptional activity of c-Jun by forming a stable heterodimer. Analysis of chimeric proteins between c-Fos and Fra-2 indicated that this difference is mainly attributable to their C terminal-half regions. Interestingly, this suppressive effect of Fra-2 was not observed in the combination with JunD: fra-2 plus junD, like c-fos plus junD, had higher transcriptional activity than junD alone. Fra-1 showed essentially the same transcriptional regulatory properties as Fra-2. These differential properties greatly expand the potential range of regulatory functions of the Fos family proteins.

Animals↗
PubMed1991

Transcription of fra-2 mRNA and phosphorylation of Fra-2 protein are stimulated by serum.

The RNA transcript and the gene product of a newly isolated fos-related gene, fra-2, were analyzed in growth-arrested and growth-stimulated chicken embryo fibroblasts (CEF). In the growth-arrested cells, a small amount of 6.8 kilobase (kb) fra-2 transcript was synthesized and several species (40-46 kilodaltons (kd) of hypophosphorylated fra-2 gene products (Fra-2) were detected. After the growth stimulation, two species of fra-2 transcripts of 6.8 kb and 5.7 kb were transiently induced. Compared with c-fos mRNA, expression of these fra-2 mRNAs was significantly prolonged, in good agreement with the previous analysis on the synthetic rate of Fos and Fra-2 proteins. Upon growth stimulation, Fra-2 undergoes more extensive phosphorylation on serine residues to form a 46 kd band, possibly owing to the protein kinase activity induced by serum.

Animals↗
PubMed1991

High-level expression of human c-jun gene causes cellular transformation of chicken embryo fibroblasts.

To analyze the transforming activity of c-jun, a Rous sarcoma virus (RSV) variant that carries human c-jun instead of v-src (JH1) was constructed. After infection onto chicken embryo fibroblasts (CEF), JH1 formed foci with a titer comparable to that of wild-type RSV, and the infected cells grew in soft agar, indicating that the human c-jun gene has transforming potential, like the v-jun gene. The expression of Fra-2, one of the recently isolated Fos-related antigens, but not Fos was detected in both JH1-infected CEF and CEF infected with the control retrovirus vector (DS3). Gel shift analysis using nuclear extracts from DS3-infected CEF revealed that the Fra-2/Jun complex contributes to the basal level of AP-1 DNA binding activity. A similar activity was detected in JH1-infected CEF, but these cells have an additional AP-1 binding activity derived from Jun homodimers that seems to play important roles in the cellular transformation.

Animals↗
PubMed1991

Inhibition of jun transformation by a mutated fos gene: design of an anti-oncogene.

The protein products of the fos and jun oncogenes (Fos and Jun) function as transcriptional regulators in the form of homo- or heterodimeric complexes that bind to DNA. Dimerization is mediated by a leucine zipper structure that serves to juxtapose alpha-helical regions of each protein, rich in basic amino acids, that form a bipartite DNA-binding domain. Although Fos participates exclusively in heterodimeric complexes, Jun can function either as a homodimer that has a low apparent affinity for DNA or as a more stable heterodimer with Fos that has a higher apparent affinity for DNA. We have used these properties of Fos and Jun to design a mutated fos gene, lacking a functional DNA-binding domain (supfos1), that suppresses the transforming activity of jun in trans. Here we show that chicken embryo fibroblasts transformed by jun revert to a normal phenotype after infection by a retroviral vector encoding supFos1. Furthermore, infection of normal cells with the supfos1 vector renders them resistant to subsequent transformation by jun. Inhibition of jun transformation was associated with the appearance of supFos1-Jun heterodimers and a reduction in the AP-1 DNA-binding activity contributed by Jun homodimers. These findings demonstrate that the function of leucine zipper-containing transcription factors can be investigated by the procedure of intracellular immunization.

Animals↗
PubMed1991

T-cell subsets in drug-induced toxic epidermal necrolysis. Possible pathogenic mechanism induced by CD8-positive T cells.

A patient with bromisovalum-induced toxic epidermal necrolysis showed pronounced delayed hypersensitivity to bromisovalum by patch testing. Biopsy specimens from the cutaneous lesion and the site of the positive patch test reaction were analyzed and compared immunohistologically. The findings were similar: most of the mononuclear cells disposed along the dermoepidermal junction and migrating into the epidermis were CD8-positive lymphocytes, whereas the dermal inflammatory infiltrates were composed predominantly of CD4-positive lymphocytes. This case showed the potential usefulness of patch testing in evaluating cases of toxic epidermal necrolysis. We believe that delayed hypersensitivity plays a crucial role in the development of drug-induced toxic epidermal necrolysis. Furthermore, potential effector cells with phenotypic characteristics of CD8-positive lymphocytes (suppressor/cytotoxic T cells) seem to represent important mediators of the epidermal damage of the cutaneous lesion in our case.

Adult↗
PubMed1990

Isolation and characterization of fra-2, an additional member of the fos gene family.

Antiserum raised against a Fos peptide (amino acids Lys-127 to Arg-140 of chicken c-Fos) recognizes a 46-kDa Fos-related protein in cell lysates of growth-stimulated chicken embryo fibroblasts. Induction of the 46-kDa protein is transient but is slightly prolonged relative to c-Fos following growth stimulation. Using a mixed oligonucleotide probe encoding the peptide antigen, we have cloned the chicken genomic locus that encodes this protein and have determined its gene structure. This locus consists of four exons, each of which has some homology with the corresponding exons of the chicken c-fos gene, and it expresses a 6-kilobase mRNA after growth stimulation. The deduced amino acid sequence of the gene (323 amino acids) contains a "leucine zipper" and includes five distinct regions that exhibit strong sequence homology to the recently identified fos-related antigens Fra-1 (rat) and FosB (mouse) as well as c-Fos. Since the other regions of the gene have little homology to any of these three proteins, this gene was named "fra-2." When the fra-2 gene was overexpressed by an avian retrovirus vector, it caused transformation of chicken embryo fibroblasts. The fra-2 gene product formed a complex in cells with the c-Jun protein, indicating that c-fos and fra-2 share biological and biochemical functions.

Amino Acid Sequence↗
PubMed1989

Identification of a small region of the v-fos gene product that is sufficient for transforming potential and growth-stimulating activity.

To analyze the structure-function relationship for the v-fos protein, we constructed in-frame insertion and deletion mutants of the v-fos gene carried by FBJ-MuSV, and expressed them in chicken primary cells using retrovirus vectors. We assessed the effects of these mutations on the ability of the v-fos protein to transform chicken embryo fibroblasts and to stimulate cellular proliferation of chicken neuroretinal cells. The mutant which retains only the central region of the v-fos protein (Met111-Ile206) have both activities, but the mutants which have deletions in this region, and one of the mutants that has a four amino acid insertion in it, lost both activities. The central region that is sufficient for these activities includes the evolutionarily highly conserved region among human, mouse and chicken c-fos proteins. Additionally, this sequence shares some homology with the DNA binding domain of GCN4 and c-jun protein. The truncated fos protein that contains only part of the central region is not phosphorylated in chicken embryo fibroblasts, indicating that phosphorylation of the fos protein is not necessary for the transforming activity.

Amino Acid Sequence↗
PubMed1988

Transforming potential and growth stimulating activity of the v-fos and c-fos genes carried by avian retrovirus vectors.

To study transforming potential as well as growth stimulating activity of the fos genes on primary cells, we have developed avian retrovirus vectors by constructing derivatives of Rous sarcoma virus DNA in which the v-src gene was replaced by either the v-fos gene of FBJ-MuSV or the mouse c-fos gene. After each derivative was introduced into chicken embryo fibroblasts by transfection, replication-competent viruses that carry the v-fos gene (FJ2) or the c-fos gene (FM4) were recovered. FM4 and FJ2 introduced the fos genes into almost all chicken embryo fibroblasts within 3 days after infection, expressed their gene products, and induced morphological transformation and colony formation in soft agar. Results show that overproduction of the c-fos gene product is enough for cellular transformation not only of rat established fibroblasts as reported previously but also of avian primary fibroblasts. Using this vector system, we have further shown that the c-fos gene and the v-fos gene have biological activities that induce cellular proliferation of chicken neuroretinal cells, which normally stay in the resting stage of growth in monolayer culture.

Animals↗
PubMed1987

The chicken c-fos gene: cloning and nucleotide sequence analysis.

Using a DNA probe from an avian transforming virus, NK24, that contains the fos gene and that was newly isolated and characterized (M. Nishizawa, N. Goto, and S. Kawai, J. Virol. 61:3733-3740, 1987), we cloned and sequenced the chicken c-fos gene. Results showed that this gene, like the mouse or human c-fos gene, contains four exons encoding a predicted gene product of 367 amino acids which is about 79% homologous to p55c-fos (mouse), excluding several small insertions or gaps. The predicted gene product, however, contains two nonhomologous regions, as suggested by sequence analysis of the NK24 genome. About half the identical amino acids of these gene products are encoded by different codons, and the guanine-plus-cytosine content in the third letter of each codon used in the entire chicken c-fos coding region is high (93%). The 5'-flanking region of the TATA box contains a sequence that is homologous to a putative transcriptional regulatory sequence in the mouse or human c-fos gene. The 3'-noncoding region of chicken c-fos is strikingly homologous to that of mouse or human c-fos and covers a 67-base-pair adenine-plus-thymine-rich stretch which was previously reported to be essential for an inhibitory effect on the expression of the mouse c-fos gene.

Amino Acid Sequence↗
PubMed1986

Activation of the transforming potential of p60c-src by a single amino acid change.

Previous work showed that overexpression of the cellular src (c-src) gene does not cause transformation of chicken cells in culture. However, viral stocks isolated from cells transfected with Rous sarcoma virus DNA containing the c-src gene in place of the viral src gene did occasionally produce foci. Virus obtained from these foci were highly transforming and appeared to arise via spontaneous mutation in the c-src-containing viral populations. The p60 proteins of the transforming mutant src viruses were found to have higher levels of in vitro tyrosine kinase activity than the levels observed with the parental viruses. In this study, we have molecularly cloned the src DNA sequences of two transforming mutant src viruses. When compared to the DNA sequence of the parental c-src viruses, the mutant viruses contain single point mutations that result in single amino acid changes in the src gene products (p60 proteins). Both amino acid changes reside in the tyrosine kinase domain of the protein. The mutation detected in one virus involves replacement of the normal Glu-378 in p60c-src by Gly, whereas the p60 of the other transforming virus has Phe instead of the normal Ile-441. Our data indicate that when p60c-src is expressed at elevated levels in a retroviral context, a single amino acid change in its primary sequence can activate the kinase activity of this protein and cause cellular transformation.

Amino Acid Sequence↗
PubMed1986

Genetic analysis of p60v-src domains involved in the induction of different cell transformation parameters.

The expression of p60v-src in chicken cells infected with Rous sarcoma virus causes stimulation of cell proliferation, morphological alteration, and anchorage independence. PA101 and PA104 are temperature-sensitive variants encoding mutant p60v-src proteins that are partially defective in the induction of these transformation parameters. To define the structural basis for the transformation defectiveness of the p60v-src mutants, the v-src genes of PA101 and PA104 were molecularly cloned and analyzed. Amino- and carboxy-terminal coding regions of the cloned mutant genes were exchanged with the corresponding regions of cloned wild-type v-src and chicken c-src genes, reconstructed into viral DNA, and expressed in infected cells maintained at various temperatures. This analysis revealed that lesions within the tyrosine kinase domains of the two mutant proteins confer temperature sensitivity on all three transformation functions of p60v-src. An amino-terminal region of the PA101 mutant protein, which coincides with the proposed modulatory domain and appears to interact with the kinase domain, affects morphological alteration in a temperature-independent manner. Our results suggest that the function of the kinase domain is essential to all three parameters examined, whereas the amino-terminal domain is important in determining cell morphology.

Amino Acid Sequence↗
PubMed1986

Biochemical properties of p60v-src mutants that induce different cell transformation parameters.

PA101 and PA104 are Rous sarcoma virus variants that are differentially temperature sensitive in cell transformation parameters, including stimulation of cell proliferation, morphological alteration, and anchorage independence. To investigate the biochemical basis for the differential expression of these parameters, the tyrosine kinase activity and subcellular localization of the mutant p60v-src proteins encoded in the variants were examined. Analysis of chimeric src proteins derived from the mutant proteins revealed that lesions in the kinase domain inhibit in vitro kinase activity and confer temperature sensitivity on tyrosine phosphorylation of cellular protein p34 in vivo. The amino-terminal portions of the mutant src proteins also influence tyrosine phosphorylation in vivo and in vitro, which is consistent with an interaction between an amino-terminal region and the kinase domain. Large proportions of the mutant src proteins exist in soluble complexes with cellular proteins p50 and p90, even though the src proteins are myristylated. The formation of these soluble complexes segregates with lesions in the kinase domain and is independent of temperature. Our results demonstrate that the transformation parameters examined correlate to a limited extent with p34 phosphorylation but not with the levels of in vitro kinase activity or soluble complex formation.

Animals↗
PubMed1986

Amino acid substitutions sufficient to convert the nontransforming p60c-src protein to a transforming protein.

We have previously shown that Rous sarcoma virus variants that carry the cellular homolog (c-src) of the viral src gene (v-src) do not transform chicken embryo fibroblasts. We also have shown that replacement of sequences upstream or downstream from the BglI site of the cellular src gene with the corresponding regions of v-src restored transforming activity to the hybrid genes. Since there are only six amino acid changes between p60c-src and p60v-src within the sequences upstream from BglI, we constructed chimeric molecules involving v-src and c-src to determine the effect of each amino acid substitution on the biological activities of the gene product. We found that the change from Thr to Ile at position 338 or the replacement of a fragment of c-src containing Gly-63, Arg-95, and Thr-96 with a corresponding fragment of v-src containing Asp-63, Trp-95, and Ile-96 converted p60c-src into a transforming protein by the criteria of focus formation, anchorage-independent growth, and tumor formation in newborn chickens. These mutations also resulted in elevation of the protein kinase activity of p60c-src.

Amino Acid Sequence↗
PubMed1985

Efficiency estimation for detecting U alpha particles in solid-state nuclear track detectors.

The detection efficiencies of solid-state nuclear track detectors, made with cellulose nitrate materials (LR-115 II) or allyl diglycol carbonate (CR-39) were investigated. Detection efficiency for a surface alpha source was experimentally obtained by changing the dimensions between the detector and the source, while alpha-particle incident efficiency was calculated. The ratio of the detection efficiency to the incident efficiency was then determined. It was confirmed that the ratio for LR-115 II was dependent on energy, but for CR-39 the ratio showed almost no dependency. Considering the relationship between solid absorber thickness and detection efficiency of the surface alpha source, detection efficiencies of U in various metals were estimated. The efficiency for U contained in Al and Fe was proposed as 16% for LR-115 II and 22% for CR-39. Using these efficiencies, amounts of U in some Al and Fe ingots were determined. These agreed with concentrations obtained by neutron-activation analysis with deviations of less than 15%.

Alpha Particles↗
PubMed1985

Lack of induction of neuroretinal cell proliferation by Rous sarcoma virus variants that carry the c-src gene.

Expression of p60v-src of Rous sarcoma virus in cultured chicken embryo neuroretinal cells was previously shown to result in the transformation and sustained proliferation of normally quiescent cell populations. We show here that Rous sarcoma virus variants that encode p60c-src, the cellular homolog of p60v-src, lack the ability to induce morphological transformation and cell proliferation of cultured neuroretinal cells. Neuroretinal cells infected with c-src-containing viruses, however, possess no less p60 protein kinase activity assayed in the immune complex than those infected with the transformation-defective Rous sarcoma virus mutants PA101 or PA104, which do stimulate the growth of these cells.

Animals↗