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M Imagawa

Publications and source records attributed to M Imagawa.

At least 19 recordsLinked to original sources
PubMed1996

Activation of glutathione transferase P gene by lead requires glutathione transferase P enhancer I.

Glutathione transferase P (GST-P) is specifically induced in rat liver and kidney by lead cation. The increase of GST-P mRNA after lead administration is blocked by actinomycin D, suggesting that GST-P production by lead is regulated at the transcriptional level. To further determine which part of the flanking region of the GST-P gene has the lead-responsive cis-element in vivo, we utilized transgenic rats with five different constructs having GST-P and/or chloramphenicol acetyl-transferase coding sequence. We studied the effect of lead on these transgenic rats and on transfected NRK (normal rat kidney) cells and found that GST-P induction by lead is indeed regulated at the transcriptional level and that the GST-P enhancer I (GPEI) enhancer is an essential cis-element required for the activation of the GST-P gene by lead. GPEI consists of two AP-1 (c-Jun/c-Fos heterodimer) site-like sequences that are palindromically arranged and can bind AP-1, c-jun mRNA in the liver increased after lead administration and GST-P, and c-Jun had patchy expression in the same hepatocytes 24 h after lead exposure. These results suggest that activation of the GST-P gene by lead is mediated in major part by enhancer GPEI and that AP-1 may be involved at least partially. GPEI has been shown to have essential sequence information for the trans-activation of the GST-P gene during chemical hepatocarcinogenesis of the rat (Morimura, S., Suzuki, T., Hochi, S., Yuki, A., Nomura, K., Kitagawa, T., Nagatsu, I., Imagawa, M., and Muramatsu, M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2065-2068; Suzuki, T., Imagawa, M., Hirabayashi, M., Yuki, A., Hisatake, K., Nomura, K., Kitagawa, T., and Muramatsu, M. (1995) Cancer Res. 55, 2651-2655). The present study establishes that the same enhancer element does operate in the activation of the GST-P gene by lead regardless of the trans-activators involved.

Animals↗
PubMed1995

CCAAT/enhancer-binding proteins alpha and beta interact with the silencer element in the promoter of glutathione S-transferase P gene during hepatocarcinogenesis.

We have previously identified a silencer in the glutathione S-transferase P (GST-P) gene which is strongly and specifically expressed during chemical hepatocarcinogenesis. At least three trans-acting factors bind to multiple cis-elements in the silencer. One of them, Silencer Factor-B (SF-B), is identical with CCAAT/enhancer-binding protein beta (C/EBP beta) and binds to GST-P Silencer 1 (GPS1). Many C/EBP beta binding sites are recognized by each of the C/EBP isoforms. Western blot analyses of C/EBP isoforms during chemical hepatocarcinogenesis revealed a decrease of C/EBP alpha expression. However, there was no change in C/EBP beta level. In the nuclear extracts from normal liver, C/EBP alpha was the dominant form that bound to GPS1, whereas both C/EBP alpha and C/EBP beta bound to GPS1 in the nuclear extracts from carcinogenic liver. Furthermore, transfection assays showed that C/EBP alpha not only repressed the GST-P promoter activity but also attenuated the transcriptional stimulation by C/EBP beta. These observations strongly suggest that the ratio of C/EBP alpha to C/EBP beta is one of the important factors for the GST-P silencer activity, and the decrease of this ratio during hepatocarcinogenesis reduces the silencer activity and, consequently, increases the GST-P expression.

Animals↗
PubMed1995

CTG triplet repeat in mouse growth inhibitory factor/metallothionein III gene promoter represses the transcriptional activity of the heterologous promoters.

Growth inhibitory factor/metallothionein III (GIF/MT-III) is expressed specifically in brain, and neither mRNA nor protein is detected in other organs. This tissue-specific expression might be regulated by negative elements as well as positive elements, such as tissue-specific enhancers. To investigate the repression mechanisms of this gene in organs other than the brain, transfection experiments were performed by using various deletion mutants. Interestingly, a 25 x CTG repeat in the promoter region seemed to contribute to the repression activity. Moreover, the repression activity of this 25 x CTG repeat was also observed in various promoters and in a direction and position independent manner, indicating that this element could act as a silencer. However, no binding protein was detected by gel-shift and footprint analyses. These results strongly suggest that the CTG repeat functions as a negative element, and that this effect is caused by unknown mechanisms, rather than by interactions between specific cis-elements and specific trans-acting factors as reported previously. It is also possible that the CTG repeat functions as a general silencer in many genes.

Alzheimer Disease↗
PubMed1995

Identification of an enhancer responsible for tumor marker gene expression by means of transgenic rats.

The glutathione transferase P (GST-P) gene is known for its specific expression during chemical hepatocarcinogenesis of the rat and is used as a tumor marker for hepatocellular carcinoma. We have shown recently that the upstream 2.9-kb region of the GST-P gene is sufficient for conferring tumor-specific expression of the gene in vivo (S. Morimura et al., Proc. Natl. Acad. Sci. USA, 90: 2065-2068, 1993). To further identify crucial sequence elements regulating the unique expression of this gene, we have established six independent lines of transgenic rats bearing distinct areas of the GST-P gene that are connected to the chloramphenicol acetyltransferase coding region and analyzed changes of the chloramphenicol acetyltransferase activity during the course of chemical hepatocarcinogenesis. We demonstrate here that the enhancer, glutathione transferase P enhancer I, that is located 2.5 kb upstream of the GST-P gene is required and sufficient for its tumor-specific expression of the gene among other controlling elements. This approach to transgene expression could be used to define other enhancers, the activity of which is dependent on cellular changes such as carcinogenesis, development, and differentiation.

Animals↗
PubMed1995

Vitamin D receptor contains multiple dimerization interfaces that are functionally different.

The vitamin D receptor mediates the signal of 1 alpha, 25-dihydroxyvitamin D3 by binding to vitamin D responsive elements in DNA as a homodimer or as a heterodimer composed of one vitamin D receptor subunit and one retinoid X receptor subunit. We have mapped the dimerization interfaces of the vitamin D receptor that is involved in homo- or heterodimer formation in the absence of DNA. While deletion of the first zinc finger region of vitamin D receptor diminished homodimerization activity, it did not affect heterodimerization. In contrast, a deletion just beyond the zinc finger region affected heterodimerization with retinoid X receptor, but not homodimerization. The zinc finger region alone could form a homodimer with full-length vitamin D receptor, but not a heterodimer with retinoid X receptor. The carboxy-terminal region was also necessary for heterodimer formation. This region showed only a weak dimerization activity in the absence of ligand, but this was dramatically increased in the presence of ligand for both homo- and heterodimerization. These results suggest that the vitamin D receptor has at least three dimerization interfaces whose functions are apparently distinguishable. These are located in the first zinc finger region, the region just beyond this zinc finger and in the carboxy-terminal region.

Amino Acid Sequence↗
PubMed1995

Axonal projections of utricular afferents to the vestibular nuclei and the abducens nucleus in cats.

Axonal projections of utricular (UT) afferents in cats were examined by three approaches: recordings of field potentials, labelling of UT nerve fibres by localized infusion of horseradish peroxidase (HRP) and intraaxonal infusion of HRP into a single UT afferent. UT afferents project principally into the rostral part of the descending nucleus and the ventral part of the lateral nucleus. Projection into the superior and the medial nuclei and the ipsilateral abducens nucleus were also observed.

Abducens Nerve↗
PubMed1995

Sequence analysis of the rat jun-D gene.

A rat jun-D genomic clone was isolated from a rat liver lambda EMBL3 library. Sequence analysis revealed a high sequence similarity with mouse jun-D, and relatively low similarity with human and chicken jun-D. In both the trans-activation and DNA-binding domains, however, the amino-acid sequences were well conserved among rat, mouse, human and chicken.

Amino Acid Sequence↗
PubMed1995

Structure of the Oct-3 POU-homeodomain in solution, as determined by triple resonance heteronuclear multidimensional NMR spectroscopy.

The POU-homeodomain (POUH) forms the bipartite DNA-binding POU domain in association with the POU-specific domain. The 1H, 15N, and 13C magnetic resonances of the 67-amino acid long POUH of mouse Oct-3 have almost completely been assigned, mainly through the combined use of three-dimensional triple resonance NMR methods. Based on the distance and dihedral angle constraints derived from the NMR data, the solution structure of the POUH domain has been calculated by the ab initio simulated annealing method. The average RMS deviation for all backbone heavy atoms of the 20 best calculated structures for residues 9-53 of the total 67 amino acid residues is 0.44 A. The POUH domain consists of three alpha-helices (helix-I, 10-20; helix-II, 28-38; and helix-III, 42-53), and helices-II and -III form a helix-turn-helix motif. In comparison with other classical homeodomains, the folding of the three helices is quite similar. However, the length of helix-III is fairly short. In the complex of the Oct-1 POU domain with an octamer site (Klemm JD, et al., 1994, Cell 77:21-32), the corresponding region is involved in helix-III. The structural difference between these two cases will be discussed.

Amino Acid Sequence↗
PubMed1995

Tissue-specific activation of tumor marker glutathione transferase P transgenes in transgenic rats.

By means of transgenic rats, we have recently shown that the GPEI enhancer of the glutathione transferase P (GST-P) gene, which has two one-base-missmatched AP-1 sites locating palindromically with three-base spacing in between, is sufficient for conferring tumor-specific activation of the gene in vivo. It is noted that there is another consensus AP-1 site near the promoter of this gene. By using seven independent transgenic rats, bearing distinct areas of the GST-P gene that are connected to the chloramphenicol acetyltransferase (CAT) coding sequence, we analyzed CAT expression in various tissues (brain, lung, liver, kidney, spleen) in these transgenic rats. We found that the ECAT gene, which has sufficient of the upstream regulatory region (approx. 2.9 kb) of the gene containing GPEI, is trans-activated in the kidney and lung of transgenic rats in a similar manner to endogenous GST-P. When either the GPEI core sequence or the AP-1 site near the promoter is deleted, CAT expression decreases to almost background level. Substitution of the GPEI core or the AP-1 site near the promoter to this silent construct (5CATGPEIcore) reconstituted CAT expression in the transgenic rats. In these rats, CAT was expressed in the brain and lung rather than in the kidney, showing a somewhat different pattern from the endogenous GST-P. In the brain tissue of the 5CATGPEIcore transgenic rat, CAT was demonstrated in the glia cells, which is consistent with endogenous GST-P expression. These results suggest that a relatively long upstream region (approx. 2.9 kb) is required for tissue-specific expression of the GST-P gene and that GST-P expression in the brain may be regulated differently from its expression in other organs.

Animals↗
PubMed1994

Difference and similarity of DNA sequence recognized by VDR homodimer and VDR/RXR heterodimer.

Nuclear receptors for the thyroid hormone and vitamin A and D cooperate with the retinoid X receptor (RXR) in activating the transcription. Although the hormone response elements for these receptors have been proposed in which spacing of the direct repeated motifs determine the specificity (so called 3-4-5 rule), vitamin D response elements (VDREs) in the natural context consist of often imperfect direct repeats. Vitamin D receptor (VDR) alone can bind to the mouse osteopontin (mSPP-1) VDRE, which contains a direct repeat separated by 3 nucleotides, but not to the rat osteocalcin (rOST) VDRE having inexact direct repeat. The presence of RXR not only allows the VDR to bind to the rOST VDRE, but also increases the binding affinity for the mSPP-1 VDRE. The RXR/VDR heterodimer exhibits the similar affinity constants for the mSPP-1 VDRE and the rOST VDRE, in spite of the apparently different affinities for two VDREs of the VDR homodimer. A random oligonucleotide selection procedure revealed that the consensus sequence selected by the RXR homodimer is the direct repeat spaced by one A residue. In contrast, the sequences preferentially selected by the VDR homodimer and the VDR/RXR heterodimer are similar, which are the direct repeats spaced by 3 nucleotides. The difference and similarity of DNA sequence recognition are discussed.

Animals↗
PubMed1994

Second-order vestibular neuron morphology of the extra-MLF anterior canal pathway in the cat.

Second-order vestibular neurons form the central links of the vestibulo-oculomotor three-neuron arcs that mediate compensatory eye movements. Most of the axons that provide for vertical vestibulo-ocular reflexes ascend in the medial longitudinal fasciculus (MLF) toward target neurons in the oculomotor and trochlear nuclei. We have now determined the morphology of individual excitatory second-order neurons of the anterior semicircular canal system that course outside the MLF to the oculomotor nucleus. The data were obtained by the intracellular horseradish peroxidase method. Cell somata of the extra-MLF anterior canal neurons were located in the superior vestibular nucleus. The main axon ascended through the deep reticular formation beneath the brachium conjunctivum to the rostral extent of the nucleus reticularis tegmenti pontis, where it crossed the midline. The main axon continued its trajectory to the caudal edge of the red nucleus from where it coursed back toward the oculomotor nucleus. Within the oculomotor nucleus, collaterals reached superior rectus and inferior oblique motoneurons. Some axon branches recrossed the midline within the oculomotor nucleus and reached the superior rectus motoneuron subdivision on that side. Since these neurons did not give off a collateral toward the spinal cord, they were classified as being of the vestibulo-oculomotor type and are thought to be involved exclusively in eye movement control. The signal content and spatial tuning characteristics of this anterior canal vestibulo-oculomotor neuron class remain to be determined.

Animals↗
PubMed1994

Acute changes in liver gene expression in the N-nitrosodiethylamine-treated rat.

We constructed a complementary DNA (cDNA) library from mRNAs of rat liver induced by an initiating dose of a chemical carcinogen, N-nitrosodiethylamine (DEN). Using a differential hybridization with cDNA probes prepared from mRNAs of control and DEN-treated rat liver, eight cDNAs of which expression was altered by an acute single dose of DEN were cloned. Colony hybridization and nucleotide sequencing demonstrated six independent cDNA clones. These were known genes encoding liver-specific proteins such as microsomal epoxide hydrolase (mEH; epoxide hydrolase, EC 3.3.2.3), albumin, transthyretin, CYP2B7, CYP1A2 (microsomal cytochrome P450, EC 1.14.14.1) and argininosuccinate synthetase (EC 6.3.4.5). Quantitative Northern blot hybridization was carried out to measure the mRNA content of DEN-initiated rat liver at various times after DEN injection. We also analyzed the expression of glutathione transferase P (GST-P; glutathione transferase, EC 2.5.1.18), c-jun and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; glyceraldehyde-phosphate dehydrogenase, EC 1.2.1.12). A single injection of DEN increased the mRNA levels of mEH, beta-actin and c-jun markedly and those of GST-P and GAPDH moderately, but decreased the mRNA levels of CYP2B7, CYP1A2, albumin and argininosuccinate synthetase. Transthyretin mRNA content was not changed, indicating that it was a false-positive clone picked up by chance. These dramatic changes in liver gene expression after acute exposure to DEN are discussed in terms of acute reactions to the massive damage to the DNA and self-defense mechanisms against toxic xenobiotics.

Animals↗
PubMed1994

Interferon as adjunctive treatment for non-metastatic renal cell carcinoma.

OBJECTIVE: To investigate the effect of recombinant alpha 2b-interferon therapy on natural killer (NK) activity and antibody-dependent cell-mediated cytotoxicity (ADCC) in the peripheral blood in patients with renal cell carcinoma who had undergone radical nephrectomy. PATIENTS AND METHODS: Between January 1988 and June 1989, 20 patients (14 men and six women, mean age 59 years, range 43-80) received interferon (IFN) therapy following radical nephrectomy for renal cell carcinoma. Thirteen patients had stage I disease, one had stage II and six had stage III; none had metastases. RESULTS: There was a significant increase in NK activity at 5 and 7 months after starting IFN therapy but there was no increase in ADCC. CONCLUSIONS: Low doses of IFN administered long-term increased NK activity in patients with renal cell carcinoma. This finding should be helpful in designating protocols for randomized studies of post-operative IFN administration as adjuvant therapy.

Adult↗
PubMed1994

Characterization of phe B gene encoding catechol 2,3-dioxygenase.

DNA sequence of the phe B gene isolated from a chromosome of the phenol degrading bacterium Pseudomonas putida BH is identical to that of the dmp B gene from the phenol degradative plasmid, pVI150. Catechol 2,3-dioxygenase encoded by phe B showed similar substrate specificity to that of xyl E. However, Phe B has much smaller Km values than Xyl E, indicating that Phe B is useful for treatment of low concentrations of catechol derivatives in waste water.

Biodegradation, Environmental↗
PubMed1994

Relaxation of rat thoracic aorta induced by the Ca(2+)-ATPase inhibitor, cyclopiazonic acid, possibly through nitric oxide formation.

1 The effect of the Ca(2+)-ATPase inhibitor, cyclopiazonic acid (CPA), was studied on rat thoracic aortic ring preparations. 2 At concentrations above 0.3 microM, CPA induced relaxation in the arteries precontracted with phenylephrine. Removal of the endothelium abolished CPA-induced relaxation. 3 The nitric oxide (NO) synthase inhibitor NG-nitro L-arginine (3-300 microM), the free radical scavenger haemoglobin (0.1-3 microM), the soluble guanylate cyclase inhibitor, LY83583 (0.1-10 microM), each inhibited the endothelium-dependent relaxation to CPA. The potassium channel blocker, glibenclamide (10 microM) and cyclo-oxygenase inhibitor, indomethacin (100 microM for 60 min and then washed out) did not alter the action of CPA. 4 The calmodulin inhibitors calmidazolium (3-10 microM) and W-7 (100 microM) also abolished CPA-induced relaxation. 5 CPA (10 microM) increased guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels in arteries with an intact endothelium, without affecting adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. 6 The inhibitors of NO synthesis and actions, the calmodulin inhibitor and removal of the endothelium abolished the CPA-stimulated increase in the levels of cyclic GMP. 7 In Ca(2+)-free solution, CPA failed to induce relaxation or to stimulate cyclic GMP production. Relaxation to nitroprusside was not affected under these conditions. 8 These results suggest that CPA can stimulate NO synthesis, possibly by inhibiting a Ca(2+)-ATPase, which replenishes Ca2+ in the intracellular storage sites in endothelial cells. Depletion of the Ca2+ store in the endothelium may then trigger influx of extracellular Ca2+, contributing to an increase in free Ca2+ in the endothelial cells, which activates NO synthase and NO formation.

Amino Acid Oxidoreductases↗
PubMed1994

Monosynaptic and disynaptic connections in the utriculo-ocular reflex arc of the cat.

1. Connections from the utricular (UT) nerve to motoneurons and interneurons in the ipsilateral abducens (AB) nucleus were studied in anesthetized and decerebrated cats. Bipolar electrodes were fixed on the left UT nerve under visual observation. The other branches of the vestibular nerve and the facial nerve were transected in the left inner ear. 2. Stimulation of the UT nerve evoked a small positive-negative (P/N) deflection and a negative (N1) potential in the vestibular nuclei, with mean latencies of 0.56 and 0.84 ms, respectively. In the AB nucleus a small P/N deflection with a mean latency of 0.72 ms was recorded, which was considered as a incoming volley of the UT nerve. 3. Excitatory postsynaptic potentials (EPSPs) were recorded from AB motoneurons with short latencies after UT nerve stimulation. They were classified into two types, M and D. M-type EPSPs, which followed repetitive high-frequency stimuli and were recorded from the majority of AB motoneurons, had latencies ranging from 0.9 to 1.2 ms. Double shocks to the UT nerve evoked EPSPs that had the same latency. It was suggested that the AB motoneurons had monosynaptic connections with the UT nerve. D-type EPSPs, which were recorded from most of the AB motoneurons, had slightly longer latencies ranging from 1.2 to 1.8 ms. They showed temporal facilitation when double shocks were provided to the UT nerve. They did not follow repetitive high-frequency stimuli (< or = 2.5-ms interval). It was suggested that D-type EPSPs were di-synaptically evoked via secondary vestibular neurons. Interneurons in the AB nucleus had the same characteristics as AB motoneurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Abducens Nerve↗