Biomedical subjects

H Ariga

Publications and source records attributed to H Ariga.

At least 55 records · Page 3Linked to original sources

PubMed1998

MM-1, a novel c-Myc-associating protein that represses transcriptional activity of c-Myc.

We have isolated the cDNA encoding a novel c-Myc-binding protein, MM-1, by the yeast two-hybrid screening of a human HeLa cell cDNA library. The protein deduced from the cDNA comprises 167 amino acids and was localized in the nucleus of introduced COS-I cells. The MM-1 mRNA was highly expressed in human pancreas and skeletal muscle and moderately in other tissues. As for the c-Myc binding, glutathione S-transferase MM-1 expressed in Escherichia coli bound in vitro to c-Myc translated in reticulocyte lysate, and almost whole, the MM-1 molecule was necessary for the binding in the yeast two-hybrid system. The mammalian two-hybrid assays in hamster CHO cells revealed that MM-1 interacts in vivo with the N-terminal domain covering the myc box 2, a transcription-activating domain, of c-Myc. Furthermore, MM-1 repressed the activation of E-box-dependent transcription by c-Myc.

Amino Acid Sequence↗

PubMed1998

Structural analysis of mouse tenascin-X: evolutionary aspects of reduplication of FNIII repeats in the tenascin gene family.

Tenascin-X (TNX) is an extracellular matrix glycoprotein involved in both primary structural functions and modulating cellular activities in multicellular organisms. We determined the 67977bp nucleotide sequence of the entire mouse tenascin-X (Tnx) gene, which also includes the last exon of Creb-rp and Cyp21. We compared it with the orthologous human locus. Conservation of both position and orientation of the three functionally unrelated genes at this position was found. Comparison also revealed that introns 1, 4 and 6 of Tnx are highly conserved between species. The sequence showed that mouse Tnx contains 43 exons separated by 42 introns. The deduced amino-acid sequence (4114 residues) revealed that mouse Tnx has a primary structure characteristic of tenascins, which consists of a signal peptide and four heptad repeats followed by 18.5 epidermal growth factor-like (EGF) repeats, 31 fibronectin type III-like (FNIII) repeats, and a region homologous to fibrinogen. cDNA clones generated by alternative splicing of eight consecutive FNIII repeats (M15-M22) as well as a proximal FNIII repeat (M3) were also identified. The FNIII motifs that were subject to alternative splicing were assigned to the group of recently reduplicated FNIII repeats because they have a high level of amino-acid sequence similarity. We also analyzed the evolution of FNIII repeats in TNX.

Amino Acid Sequence↗

PubMed1998

Characterization of an element positively regulating the transcription of MSSP gene-2 which encodes C-MYC binding proteins.

MSSP gene-2 encodes at least three alternative splicing products, MSSP-1, MSSP-2, and Scr2, which have been implied to function as factors regulating DNA replication, transcription, apoptosis induction, and cell-cycle movement, via the interaction with C-MYC. We have previously shown that the Mup1 sequence from -474 to -440 in the region required for transactivation of the MSSP gene-2 was protected from DNaseI digestion in vitro. Here, we report that the Mup1 sequence carries a positive transcriptional activity, and that the activity was lost by point mutations that inhibited the formation of specific nucleoprotein complexes on the sequence. In Southwestern analyses, two proteins of 38 and 62kDa directly interacted with the Mup1 sequence.

Alternative Splicing↗

PubMed1998

c-Myc gene single-strand binding protein-1, MSSP-1, suppresses transcription of alpha-smooth muscle actin gene in chicken visceral smooth muscle cells.

The expression of alpha-smooth muscle actin is coordinately regulated by positive and negative cis- elements in the promoter region. Although cis -elements and trans -acting factors involved in the positive regulation of the alpha-smooth muscle (alpha-SM) actin gene have been well characterized, details of negative regulation remain unclear. In functional analyses using cultured gizzard smooth muscle cells, we identified a sequence ranging from -238 to -219 in the promoter region as a novel negative element. Mutation and deletion analyses further revealed that a sequence, TATCTTA (-228 to -222), is essential for negative regulation. Gel shift assay and Southwestern blotting indicated that a nuclear protein factor specifically interacts with single- or double-strand DNA including this sequence, and the protein factor displays a highly potent binding to the sense strand DNA. cDNA cloning and gel shift analysis using anti-MSSP-1 antibodies revealed that this protein factor is a chicken homolog of human MSSP-1 (c- myc gene single-strand binding protein-1). In fact, overexpression of MSSP-1 in cultured smooth muscle cells suppresses the promoter activity. These results suggest a novel function of MSSP-1 regarding the transcriptional regulation of alpha-sm actin gene.

Actins↗

PubMed1998

AMY-1, a novel C-MYC binding protein that stimulates transcription activity of C-MYC.

BACKGROUND: The c-myc proto-oncogene has been suggested to play key roles in cell proliferation, differentiation, transformation and apoptosis. A variety of functions of C-MYC, the product of c-myc, are attributed to protein-protein interactions with various cellular factors including Max, YY1, p107, Bin1 and TBP. Max and YY1 bind to the C-terminal region of C-MYC, while p107, Bin1 and TBP bind to the N-terminal region covering myc boxes. The N-terminal region is involved in all the biological functions of C-MYC, and different proteins are therefore thought to interact with the N-terminal region of C-MYC to display different functions. RESULTS: We cloned two cDNAs which encode a novel C-MYC-binding protein of 11 kDa, designated AMY-1 (Associate of C-MYC). The two cDNAs, AMY-1L and AMY-1S, derived from alternative usage of polyadenylation signals, code for the same protein of 11 kDa. AMY-1 was bound via its C-terminal region to the N-terminal region of C-MYC (amino acids nos 58-148) corresponding to the transactivation domain. AMY-1 was localized in the cytoplasm in cells expressing c-myc at low levels, but in the nucleus in the cells of a high c-myc expression in transiently transfected cells. A similar difference in endogenous AMY-1 localization was observed during the cell cycle: AMY-1 translocated from cytoplasm to nucleus during the S phase when c-myc expression was increased. AMY-1 by itself did not recognize the E-box element, the MYC/Max binding sequence, nor did it transactivate via the element, but stimulated the activation of E-box-regulated transcription by MYC/Max. FISH analyses revealed that the amy-1 gene was located at 1p32.2-1p33 in human genome. CONCLUSIONS: AMY-1 is a 11 kDa protein which binds to the N-terminal region of C-MYC and stimulates the activation of E-box-dependent transcription by C-MYC. AMY-1, which mostly localizes in the cytoplasm, translocates into the nucleus in the S phase of the cell cycle upon an increase of c-myc expression, and may thus control the transcriptional activity of C-MYC.

Amino Acid Sequence↗

PubMed1998

Fatal hemorrhage in irradiated esophageal cancer patients.

Between 1980 and 1994, 423 patients with esophageal cancer were given curative radiation therapy. Of these patients, 31 died of massive hemorrhage and were used as the subjects of analysis in this study. The incidence of massive hemorrhage in all patients was 7% (31/423). In the 31 patients who died of massive hemorrhage, 27 had local tumors and two had no tumors at hemorrhage (two unknown cases). The mean time interval from the start of radiation to hemorrhage was 9.2 months. In 9 autopsy cases the origin of hemorrhage was a tear of the aorta in 5 cases, necrotic local tumor in 3 cases and esophageal ulcer in 1 case. The positive risk factors for this complication seemed to be excess total dose, infection, metallic stent, and tracheoesophageal fistula. Chest pain or sentinel hemorrhage proceeding to massive hemorrhage was observed in about half of the patients.

Aged↗

PubMed1998

Ku antigen binds to Alu family DNA.

The GC-rich segment containing GGAGGC (Alu core) is conserved within the RNA polymerase III (pol III) promoters of Alu family sequences. We have shown that the GGAGGC motif functions as a modulator of DNA replication as well as of transcription, and identified the proteins binding to the motif in human HeLa cells. In this study, the Alu core binding proteins were partially purified from human Raji cells by using an Alu core DNA affinity column. Both the proteins thus purified were implied to be subunits of Ku antigen based on the following criteria: The molecular weights of the proteins estimated on gel electrophoreses were 70 and 85 kDa, respectively, under denaturing conditions, while under non-denaturing conditions only one band was observed for the same sample at 150 kDa, probably representing hetero-dimer formed between the 70 and 85 kDa proteins. The sizes and the hetero-dimer formation are reminiscent of the 70 and 80 kDa subunits of Ku antigen (Ku-p70 and Ku-p80). Moreover, the purified proteins were immunoreactive with anti-Ku antibodies, and the specific DNA-protein complex on the Alu core element was cancelled by the anti-Ku antibodies. The nucleoprotein complex showed the same clipping patterns as those of the complex between the Alu core element and an authentically purified Ku antigen after proteolytic cleavage with trypsin and chymotrypsin.

Antigens, Nuclear↗

PubMed1997

Structural characterisation of the exocellular polysaccharide produced by Streptococcus thermophilus OR 901.

The exocellular polysaccharide of Streptococcus thermophilus OR 901, isolated from partially deproteinised whey, is a heteropolymer of D-galactopyranose and L-rhamnopyranose residues in the molar ratio 5:2. The structure was established by methylation analysis and 1D and 2D NMR spectroscopy of the native polysaccharide, in combination with characterisation of oligosaccharide fragments, obtained by partial acid hydrolysis, using methylation analysis and 1D 1H NMR spectroscopy. The polysaccharide has a branched heptasaccharide repeating unit with the following structure: [sequence: see text]

Carbohydrate Conformation↗

PubMed1997

A novel G1-specific enhancer identified in the human heat shock protein 70 gene.

Expression of the human heat shock protein 70 gene (hsp70) is induced by various kinds of stress and by oncogenes. In the absence of stress, hsp70 is mainly expressed in the G1and S phases of the cell cycle, but the elements contributing to cell cycle-dependent expression from the hsp70 promoter remain elusive. We have previously reported that two elements, named HSP-MYCA and HSP-MYCB, located approximately 200 bp upstream (-200) from the transcription start site (+1) of human hsp70, are important for initiation of DNA replication at the hsp70 locus. In this report we examine the effect of these two elements on transcriptional activity from the hsp70 promoter, especially in terms of cell cycle-dependent expression. Various segments of the hsp70 promoter region (up to -300) were linked to the luciferase gene and the constructs were transfected into mouse L cells to examine their transcriptional activity. A strong enhancer activity was defined in the HSP-MYCB element, but not in HSP-MYCA. Mutations introduced within HSP-MYCB abolished the transcriptional activation. In synchronized cells, pHB-Luc (a luciferase construct containing approximately 2.4 kb of the hsp70 promoter region) as well as endogenous hsp70 showed two peaks of expression; one in G1 and the other in the S phase. Site-directed mutagenesis of HSP-MYCB in pHB-Luc abolished the expression peak in G1, but not that in the S phase. To test promoter specificity, wild-type and mutant HSP-MYCB elements were then linked to the luciferase gene in combination with the hsp70 , the cyclin A or the PCNA promoter. Both in transient experiments and established cell lines, a strong peak of expression in mid-G1phase was observed with all the constructs containing wild-type HSP-MYCB, but not with the constructs containing the mutant sequence. These results suggest that the HSP-MYCB sequence is a G1-specific enhancer and is responsible for cell cycle-dependent expression of hsp70.

3T3 Cells↗

PubMed1997

Cross-family interaction between the bHLHZip USF and bZip Fra1 proteins results in down-regulation of AP1 activity.

Heterodimerization among the basic-leucine zipper (bZIP) proteins or among the basic-helix-loop-helix-leucine zipper (bHLHZip) proteins confers a multitude of combinational activities to these transcription factors. To further examine the function of the bHLHZip protein, USF, we screened for cellular proteins which could directly interact with USF using the yeast two-hybrid system. A bZip protein, Fra1, was found to efficiently interact with USF. USF specifically interacts with Fra1 but not with other closely related family members, c-Fos, Fra2, FosB, or with c-Jun. Both the bHLHZip and the N-terminal regions of Fra1 are required for efficient interaction with USF. In vivo association between USF and Fra1 has been demonstrated by co-immunoprecipitation. Expression of exogenous USF led to a decrease in AP1-dependent transcription in F9 cells. Co-expression of exogenous Fra1 restored the AP1 activity in a dose-dependent manner. These data show that USF and Fra1 physically and functionally interact demonstrating that cross-talk occurs between factors of distantly related transcription families.

Animals↗

PubMed1997

DJ-1, a novel oncogene which transforms mouse NIH3T3 cells in cooperation with ras.

We have isolated and characterized the cDNA encoding a novel protein designated DJ-1. DJ-1, sharing no significant homology with the sequences so far reported, did not show transactivation activity in the Gal4 recombinant system, but transformed mouse NIH3T3 cells by itself. Furthermore, DJ-1 showed a cooperative transforming activity with H-Ras, more than 3 times as strong as the activity of ras/myc combination. DJ-1 was ubiquitously expressed in various human tissues, and the expression was induced by growth stimuli. Moreover, DJ-1 translocated from cytoplasm to nuclei in the S phase of the cell cycle. DJ-1 is thus suggested to be a novel mitogen-dependent oncogene product involved in a Ras-related signal transduction pathway.

3T3 Cells↗

PubMed1997

Human mucus protease inhibitor in airway fluids is a potential defensive compound against infection with influenza A and Sendai viruses.

Tryptase Clara, a trypsin-like protease localized exclusively in and secreted from Clara cells to the bronchial epithelium of rat, proteolytically activates the infectivity of influenza A virus [H. Kido, Y. Yokogoshi, K. Sakai, M. Tashiro, Y. Kishino, A. Fukutomi, and N. Katunuma (1992) J. Biol. Chem. 267, 13573-13579]. We report here that human mucus protease inhibitor (MPI), a major inhibitor of granulocyte elastase in the lining fluids of the human respiratory tract, significantly inhibited proteolytic activation of the infectivity of influenza A and Sendai viruses by tryptase Clara in vitro and multi-cycles of mouse-adapted influenza A virus replication in rat lungs in vitro. Recombinant MPI and the C- but not the N-terminal domain of the MPI inhibited both the proteolytic activity of tryptase Clara and the activation of virus infection. The 50% inhibitory concentrations of recombinant MPI and the C-terminal domain for tryptase Clara with Sendai virus envelope glycoprotein as substrate were 7.4 and 61.6 nM, respectively. These results indicate that MPI is a defensive compound against virus infection. Since there is evidence suggesting that concentrations of MPI in respiratory fluids are insufficient for prevention of virus infection, administration of MPI in the airway may be useful for treatment of these virus infections.

Animals↗

PubMed1997

Induction of apoptosis in HeLa cells by MSSP, c-myc binding proteins.

MSSP (c-myc gene single strand binding proteins) were identified as protein factors binding to a putative replication origin/transcriptional enhancer sequence present upstream from the human c-myc gene, and two cDNAs encoding highly homologous proteins, MSSP-1 and MSSP-2, have been cloned. Scr2, independently cloned as a factor which complements the cdc2 defective mutant of Schizosaccharomyces pombe, has turned out to be identical to MSSP-1. MSSP-1/Scr2 and MSSP-2 similarly stimulated the initiation of SV40 DNA replication, and thus were suggested to be involved in regulation of cell cycle movement, especially from the G1 to S phase. Here, we examined the functions of MSSP in apoptosis. MSSP expression plasmids were transfected to human HeLa cells together with a beta-galactosidase expression vector. After incubation in the presence of 2% calf serum, cells were stained with X-gal and morphologically apoptotic cells among the beta-galactosidase-positive cells were counted. Both MSSP-1 and 2 induced apoptosis in a dose-dependent manner as in the control experiments with c-myc or adenovirus E1A. DNA fragmentation, a hallmark of apoptosis, was also observed in cells transfected with MSSP expression plasmids. The results of experiments using various deletion mutants of MSSP indicated that the region containing one of the two RNP consensus motifs, RNP1-B, is required for induction of apoptosis as well as specific DNA binding activity.

Adenovirus E1A Proteins↗

PubMed1997

Effect of transcriptional regulatory sequences on autonomous replication of plasmids in transient mammalian systems.

Transcriptional regulatory sequences often influence the efficiency of DNA replication, directly or indirectly, in bacteria, yeast, and animal virus systems. We have tested several transcriptional regulatory sequences for affecting DNA replication, based on pUC vector, in transient systems. Autonomous replication of transfected plasmids was assayed by PCR amplification of the fragments derived from the plasmids, which had replicated in mammalian cells. By this highly efficient method of detecting replicated molecules, pUC19, but not pUC18, showed a week replication activity in transfected cells. Nucleotide sequences around the HindIII site in pUC19 were required for replication. Monomers or dimers of the octamer transcription motif of the mouse immunoglobulin heavy chain gene, inserted in multicloning sites of pUC19, could stimulate replication, while the 4- or 6-mers did not, in contrast to the results on its transcription activity. Other transcriptional elements including AP1, HSE, and E2F also stimulated replication, but neither CRE nor Sp1 binding motif did. These results suggest that at least some of the transcriptional regulatory sequences function as-modulators of DNA replication as well as of transcription.

Animals↗

PubMed1997

Determination of the functional domain of a mouse autonomous replicating sequence.

We previously isolated from mouse cells an autonomous replicating sequence (ARS) ARS65 (Ariga, Itani and Iguchi-Ariga, Mol. Cell. Biol. 7, 1-6, 1987). Here we report the nucleotide sequence of ARS65. The sequence from BgIII to EcoRI sites cloned as ARS was 2658 bp long. There exist three interesting domains: a TA repeat, a myc like box (essential sequence for c-myc ARS), and a T rich region. Cloned DNAs containing various segments of pARS65 were transfected to rat 3Y1 cells together with the hygromycinB resistance expression vector, and hygromycinB resistant clones were isolated. Established cell lines transfected with plasmids carrying either a myc-like box or a T rich region harbored the replicated plasmids, indicating that these two elements are necessary for the ARS function of pARS65.

Animals↗

PubMed1997

[Radiation therapy for low-grade astrocytomas: survival and QOL].

From 1980 to 1994, 59 patients with a diagnosis of lowgrade astrocytoma were treated in our hospital. We analyzed survival, prognostic factors and quality of life (QOL) in survivors who had been recurrence free for at least 2 years. The overall 2-, 5- and 10-year survival rates were 75, 65 and 49 % respectively. The major prognostic factors were field size (the smaller, the better) and age (the younger, the better) according to Cox regression analysis. Quality of life was evaluated in the 20 patients who had survived at least 2 years without tumor regrowth. Performance status was good in most of the patients, and 17(85%) patients were intellectually and physically normal. Headache, fatigue and memory difficulties were the major clinical complaints of these patients and were observed in 7(35%), 6(30%) and 7(35%) of the patients, respectively, although severe symptoms were rare.

Adolescent↗

PubMed1997

[Effects of combination chemoradiotherapy with daily low-dose CDDP for esophageal cancer--results of a randomized trial].

Between 1994 and 1996, 24 patients with esophageal carcinoma were entered in a randomized clinical trial comparing radiotherapy (RT) only (arm A) versus RT and a daily low dose of CDDP (5 mg/m2) (arm B). For arm B group, CDDP was injected within 30 min after irradiation. All patients were irradiated with a total dose of 60 Gy /30fr. at first, and some patients were boosted. An overall response rate of 75.0% (complete response rate of 25.0%) was observed in arm A and 91.7% (complete response rate of 16.7%) in arm B, respectively. The survival rate at 1 year was 23.8% in arm A and 40.0% in arm B. Median survival time was 7 months in arm A and 9 months in arm B. Toxicity was acceptable and no significant difference was noted between arm A and arm B.

Aged↗

PubMed1997

[Isolation and analysis of two cDNA-clones expressing the human lymphocyte expression library, inducing DNA-binding activity in vitro].

Two cDNA clones are sequenced which were isolated from human lymphocyte expression library using Southwestern (DNA-binding) screening with 32P-labeled Alu DNA in the presence of 100-fold excess of unbalanced poly (dI-dC). In one of the sequenced clones (vb22) an open reading frame (ORF) is detected, encoding protein with a new potential DNA-binding (zink-finger) domain, and DNA-binding activity of the protein is directly confirmed after its expression (as GST-fusion protein) in Escherichia coli. The other sequenced clone (wa12) is partially homologous to 15EST sequences present in GENBANK (April, 1996) and cloned from very different human tissues. Connection of these 15 overlapping GENBANK sequences resulted in a longer sequence covering wa12 and having ORF potentially encoding a new 10 kDa polypeptide without any apparent DNA-binding domains. This connected sequence as well as wa12 sequence having only 65 amino acids ORF are unrecognizable by computer software as the protein-coding regions, and we suppose that wa12 transcripts possess DNA-binding activity. Homopyrimidine blocks in RNA longer than 12 nucleotides are known to bind mirror duplex DNA sequences to form triplexes whose stability is comparable to that of protein-DNA complexes, and human promoters contain many such blocks.

Amino Acid Sequence↗