Biomedical subjects

R Tokunaga

Publications and source records attributed to R Tokunaga.

At least 37 records · Page 2Linked to original sources

PubMed1997

Involvement of the transcriptional factor GATA-1 in regulation of expression of coproporphyrinogen oxidase in mouse erythroleukemia cells.

Coproporphyrinogen oxidase (CPO; EC 1.3.3.3), the sixth enzyme of heme biosynthesis, transcribed from a single promoter is markedly induced during erythroid differentiation. CPO is ubiquitously expressed in all cells. To determine cis-acting elements of the human CPO gene, the promoter region of the gene was isolated, and three potential GATA-1 motifs and four GC boxes were found within this fragment. In a functional analysis of various deletion mutants, we found that the GATA-1 binding site at -143 to -138 was essential for basic and inducible expressions of the CPO gene in mouse erythroleukemia (MEL) cells. Gel mobility shift assay revealed that GATA-1 bound to the region is required for the expression and this was confirmed by observations that the nuclear protein bound to the GATA-1 motif was supershifted with anti GATA-1 antibody, by gel mobility shift assay. Furthermore, co-expression of mouse GATA-1 in MEL cells led to an increase in the promoter activity, which was markedly increased by dimethyl sulfoxide-treatment. These results indicate that GATA-1 plays an important role in regulation of transcription of the CPO gene in erythroid cells.

Animals↗

PubMed1997

Regulation of the ferrochelatase gene expression during differentiation of mouse erythroleukemia cells.

Ferrochelatase [EC 4.99.1.1], the last step enzyme of heme biosynthesis, is transcribed from a single promoter. The enzyme is ubiquitously expressed in all cells, while the transcription is induced during erythroid differentiation. In a transient transfection assay, we identified two cis-acting elements involved in regulating the human ferrochelatase gene in mouse erythroleukemia (MEL) cells; one is the distal region (-108 to -95) for the basic expression in erythroid and non-erythroid cells, and the other is the proximal region (-80 to -72) corresponding to the induced expression during differentiation of dimethylsulfoxide-treated MEL cells. DNase I footprinting and gel mobility shift analyses revealed the presence of nuclear protein binding to the distal and the proximal regions, in both induced and uninduced MEL cells. These mean that two promoter regions play an important role in regulating the induced expression of ferrochelatase.

Animals↗

PubMed1997

Work and workload of nursing personnel in a nursery school and two institutions for handicapped children.

Work-related low back pain and cervicobrachial disorders have become increasingly common among nursing personnel with the increase in the number of social welfare institutions and workers. The aim of this study is to assess the burden on the low backs of personnel caring for children. We measured work content, working time, posture and muscular activity in 16 nursery workers in three different types of institutions for healthy and handicapped children. The working time and posture of 7 nursery teachers in a public nursery school for healthy children (A school), 7 nursing workers (4 nursery teachers and 3 nursery instructors) in a public daycare center for mentally and physically handicapped children (B center), and 2 nursery instructors in a private home for severely handicapped children (C home) were analyzed. The muscular activity of the erectores spinae muscle of three subjects was measured using a newly developed EMG monitor (portable EMG monitor, PEMM). The following results were obtained. The average length of a lesson or activity in A school was greater than that in B center and C home. The time required by care for meals was longest in C home. The arrangement and rearrangement times were longest in the classes of children aged 3-5 in A school. The time occupied for clerical work and other work was relatively longer, while recess including lunch time was approximately one hour. The most commonly observed posture was sitting on the floor in all kinds of classes, with this occurring most often in C home. Hazardous postures such as bending forward, squatting, and kneeling appeared in 11-18% (48-81 min) of a day's working time. The average muscular activity of the erectores spinae muscle of three subjects throughout a day was approximately 10% of the maximum isometric contraction (% maximum work load, %MWL), when muscular activities were measured by PEMM. The relatively high %MWL values (19-27%MWL) were obtained at a slightly bending posture and a kneeling posture, while lower (1-9%MWL) values were observed at a sitting posture. The %MWL values at a sitting posture varied according to such conditions as duration of sitting, back rest, weight on the arms, twisting of the trunk, and hip movement. The risky nature of the sitting posture are also discussed.

Adult↗

PubMed1996

Requirement of multiple DNA-protein interactions for inducible expression of RNR3 gene in Saccharomyces cerevisiae in response to DNA damage.

The RNR3 gene encodes the large subunit of ribonucleotide reductase. Transcription of this gene is induced 12-fold in response to DNA damage or by a DNA replication blocker. To investigate cis-acting regulation, deletion analysis of the promoter region of the RNR3 gene was performed and we identified two upstream-repressing sequences in the RNR3 regulatory region. An 18-base-pairs fragment, termed DNA-damage responsive element 1 (DRE1) located between -212 and -194 in this region was found to be essential for the induction of RNR3. This fragment contained a negatively acting sequence where a protein factor bound to the region during normal growth but disappeared by exposure to 4-nitroquinoline-1-oxide. The other repressive element homologue to DRE1 was located at -263 to -254. One possible upstream-activating sequence which regulates the basal expression of RNR3 was also found. These results show that at least three potential cis-elements are necessary for the inducible expression of yeast expression of yeast RNR3 in response to DNA damage.

4-Nitroquinoline-1-oxide↗

PubMed1996

Mouse coproporphyrinogen oxidase is a copper-containing enzyme: expression in Escherichia coli and site-directed mutagenesis.

We previously isolated cDNA for mouse coproporphyrinogen oxidase (CPO) and provided evidence for the induction of mRNA during differentiation of murine erythroleukemia cells (Kohno et al. (1993) J. Biol. Chem. 268, 21359-21363). To better understand the structure and the mechanisms of reaction of the enzyme, we expressed mouse CPO in Escherichia. coli and purified it to a homogeneity. Analysis of the metal content revealed that the recombinant mouse CPO contains one copper atom per polypeptide chain. When the bacterial cells were treated with D-penicillamine, a copper chelator, formation of the active CPO was partially reduced. Addition of Cu2+ in minimal medium resulted in 6-fold higher level of CPO activity. These results suggest that expression of active mouse CPO in E. coli depended on the presence of Cu2+ in the culture medium. To elucidate the apparent involvement of Cu2+ in enzyme function, a series of mutant enzymes, whose highly conserved histidine and cysteine residues were individually converted to alanine residue, were prepared by site-directed mutagenesis. Mutant enzymes were expressed in E. coli and their activities examined. Mutation at histidine 158 resulted in a complete loss of enzyme activity, yet the enzyme protein was expressed at a comparable level. Concomitantly, only a trace amount of Cu2+ was detected in the purified H158A enzyme. We propose that mouse CPO is copper-containing enzyme and Cu2+ interacts with a conserved histidine residue.

Amino Acid Sequence↗

PubMed1996

Induction of peripheral-type benzodiazepine receptors in mouse brain following thioacetamide-induced acute liver failure.

To investigate the possible role of peripheral-type benzodiazepine receptors (PBR) in hepatic encephalopathy, we examined expression of PBR in mouse brain following thioacetamide (TAA)-induced acute liver failure. Treatment of mice with TAA resulted in an increase in the number of binding sites of the PBR ligand [3H] Ro5-4864 to brain homogenates, with no significant change in affinity of the ligand. The order of potency of different ligands to compete against [3H] Ro5-4864 binding in the brain of TAA-treated mice was Ro5-4864 > PK11195 > diazepam > protoporphyrin IX, findings similar to those in the control. Northern blot analysis revealed an increase in PBR/isoquinoline binding protein (PBR/IBP) mRNA in mouse brain following TAA treatment, in a time- and dose-dependent manner. These results indicate that the increased number of PBR in the brains of TAA-treated mice relates to the induction of PBR/IBP expression and suggest that the induction of PBR in brain may contribute to pathogenesis of hepatic encephalopathy.

Animals↗

PubMed1996

Human trehalase: characterization, localization, and its increase in urine by renal proximal tubular damage.

By using polymerase chain reaction, cDNA encoding human renal trehalase has been isolated. The partial amino acid sequence deduced by the cDNA showed homologies in rabbit, Tenebrio molitor and silkworm trehalase. Northern blots showed renal trehalase mRNA to be about 2.0 kb. To examine the properties of renal and urinary human trehalase, the trehalase cDNA was inserted in the pMAL-cRI vector downstream from the malE gene, which encodes maltose-binding protein. Transfection of the recombinant pMAL-cRI in Escherichia coli provided high levels of expression of the maltose binding protein-trehalase fusion protein. A rabbit was immunized with purified fusion protein, and antihuman trehalase antibodies were obtained. Immunoblot analysis disclosed that renal and urinary trehalase exhibited a molecular mass of about 75 kDa. Analysis by indirect fluorescent microscopy demonstrated that the enzyme located in only proximal tubular cells. Urinary trehalase activity was low in the healthy infants and elevated in patients with asphyxia. Markedly high activity was observed in a patient with Lowe syndrome. The immunoreactive urinary trehalase with 75 kDa was increased dependent on the elevation of the activity. On the basis of these findings, we conclude that the increase of urinary trehalase reflects the extent of renal tubular damage, and we propose that urinary trehalase can be a specific marker of renal tubular damage.

Amino Acid Sequence↗

PubMed1995

Induction in the gene RNR3 in Saccharomyces cerevisiae upon exposure to different agents related to carcinogenesis.

The induction of the gene RNR3 was investigated in yeast Saccharomyces cerevisiae using RNR31 lacZ fusion. Gene induction was monitored by measuring beta-galactosidase activity. Various drugs that cause DNA damage effectively induced RNR3 expression; alkylating agents (cisplatin, mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine), a radical producer (bleomycin), and an intercalator (actinomycin D) induced RNR3. When yeast expressing rat CYP1A1 was exposed to 2-aminofluorene, a concentration-dependent induction of RNR3 was observed. Aflatoxin B1 also induced the expression of RNR3 in the same yeast strain concomitant with inhibition of cell growth. In control yeast, no induction of RNR3 was observed upon exposure to 2-aminofluorene or aflatoxin B1. Exposure to 2-acetylaminofluorene or benzo[a]pyrene did not lead to induction of RNR3 in yeast expressing CYP1A1. These results indicate that DNA damage by chemicals related to carcinogenesis induces RNR3, and that activation of these procarcinogens was required for DNA damage-dependent induction of RNR3.

Animals↗

PubMed1995

The human protoporphyrinogen oxidase gene (PPOX): organization and location to chromosome 1.

We determined the structure of the human protoporphyrinogen oxidase (PPOX) gene after isolation and characterization of lambda phage clones mapping discrete regions of the cDNA. Southern blotting of human genomic DNA showed that there is a single copy of the PPOX gene, and fluorescence in situ hybridization to metaphase chromosomes mapped the gene to region 1q22. The gene has 13 exons and about 8 kb. The exon/intron boundary sequences conform to consensus acceptor (GTn) and donor (nAG) sequences, and exons in the gene appear to encode functional protein domains. Primer extension analysis revealed two major transcriptional initiation sites in a region with sequence motifs characteristic of a promoter. The promoter region contains multiple Sp1 elements, CCAAT boxes, and potential GATA-1 binding sites. Mapping of the 5' end PPOX mRNA by polymerase chain reaction indicated that there are the same transcripts in erythroid and nonerythroid cells.

Bacteriophage lambda↗

PubMed1995

Nitric oxide-mediated inactivation of mammalian ferrochelatase in vivo and in vitro: possible involvement of the iron-sulphur cluster of the enzyme.

To investigate the role of the iron-sulphur cluster in mammalian ferrochelatases, the terminal enzyme of the haem biosynthetic pathway, we examined the interaction of nitric oxide (NO) and ferrochelatase. When macrophage cell line RAW 264.7 cells were treated with interferon-gamma and lipopolysaccharide NO synthesis in the cells was stimulated, and a decrease in ferrochelatase activity was observed, with no change in the amount of ferrochelatase. The addition of NG-monomethyl-L-arginine, a selective inhibitor of NO synthesis, reduced the effect of interferon-gamma and lipopolysaccharide, while the effect of NG-monomethyl-L-arginine was suppressed by the addition of L-arginine, a substrate of NO synthase. When purified recombinant human ferrochelatase was treated with 3-morpholinosydnonimine, a NO-generating compound, ferrochelatase activity decreased with disappearance of characteristic absorbance spectra of the iron-sulphur cluster. S-Nitroso-N-acetylpenicillamine also reduced the activity, in a dose-dependent manner. These results indicate that ferrochelatase activity can be modulated by NO synthesis probably through disruption of the iron-sulphur cluster. We propose that inactivation of ferrochelatase mediated by NO (or NO-derived species) may play a role in the regulation of haem metabolism.

Amino Acid Oxidoreductases↗

PubMed1995

Formation of 4,4'-methylene-bis(2-chloroaniline)-DNA adducts in yeast expressing recombinant cytochrome P450s.

N-Oxidation of 4,4'-methylene-bis(2-chloroaniline) (MBOCA) may lead to formation of DNA adducts. To determine if cytochrome P450s are involved in the formation of MBOCA derived-DNA adducts, yeast strains expressing rodent P450s were exposed to MBOCA, and 32P-postlabelling of nucleotides from yeast genomic DNA was done. Chromatographic analysis on PEI cellulose showed that, upon exposure to MBOCA for 1 h, nine DNA adducts were formed in yeast expressing phenobarbital-inducible rabbit P450 2B5. With a 4-h-exposure, all adducts increased in parallel. In cell-free experiments, the incubation of MBOCA with phenobarbital-induced rat microsomal fraction followed by incubation with thymus DNA, led to the formation of more than ten DNA adducts. When yeast expressing 3-methylcholanthrene-inducible rat P450 1A1 was exposed to MBOCA, one major and two minor adducts were formed. No adducts were detected in control yeast. These results show that recombinant rabbit P450 2B5 exhibits a potential activation of MBOCA and that rat P450 1A1 has some effect. The use of yeast expressing recombinant P450s and the technique of 32P-postlabelling facilitates a simple search for chemicals with carcinogenic potential.

Cytochrome P-450 Enzyme System↗

PubMed1995

Induction of terminal enzymes for heme biosynthesis during differentiation of mouse erythroleukemia cells.

To examine the induction of terminal enzymes of the heme-biosynthetic pathway during erythroid differentiation, mouse protoporphyrinogen oxidase (PPO) cDNA has been cloned. The deduced amino acid sequence derived from the nucleotide sequence revealed that mouse PPO consists of 477 amino acid residues, without the leader peptide, which is imported into mitochondria. Comparison of the amino terminus of the deduced amino acid sequence of mouse PPO cDNA with that of purified bovine PPO provided conclusive evidence for lack of the leader peptide in the former. The amino acid sequence has 86% and 28% identity with human PPO and Bacillus subtilis HemY, respectively. When mouse erythroleukemia (MEL) cells were induced with dimethylsulfoxide, PPO mRNA was induced within 12 h of treatment, and with further incubation, reached a plateau. mRNAs for coproporphyrinogen oxidase (CPO) and ferrochelatase (FEC) were induced within 12 h, and continued to increase with time up to 48 h. The activities of CPO and FEC markedly increased with time up to 72 h, while PPO activity increased 1.8-fold within 12 h and remained unchanged thereafter. Immunoblot analysis showed that levels of PPO, CPO and FEC paralleled their corresponding activities. The magnitude of PPO induction was less than that of CPO and FEC. Thus, induction of three terminal enzymes of the heme-biosynthetic pathway is an early event in MEL cell differentiation. The concomitant induction may play an important role in producing large amounts of heme during erythroid differentiation.

Amino Acid Sequence↗

PubMed1995

Abnormalities of B cells and dendritic cells in SAMP1 mice.

The age-related changes in the function of antigen-presenting cells (APC) were examined using a substrain of senescence-accelerated mouse (SAMP1). In the primary mixed lymphocyte reaction (MLR), dendritic cells (DC) from aged SAMP1 mice showed less stimulatory activity than those of age-matched BALB/c or young SAMP1 mice. In the secondary MLR, the stimulatory activity of B cells was found to be lower in aged SAMP1 mice but not in age-matched BALB/c or young SAMP1 mice. In addition, these age-related decreases in the stimulatory activity of APC were found to be related to changes in the surface density of major histocompatibility complex class II and intercellular adhesion molecule-1 (ICAM-1) (but not B7-1 or B7-2 molecule) on APC (DC and B cells).

Aging↗

PubMed1995

Involvement of peripheral-type benzodiazepine receptors in the intracellular transport of heme and porphyrins.

To investigate the involvement of peripheral-type benzodiazepine receptors (PBR) in heme metabolism, we examined the interaction of [55Fe]heme with PBR. Transfection of the cloned mouse PBR-isoquinoline carboxamide-binding protein (PBR/IBP) cDNA into monkey kidney Cos-1 cells resulted in a 2.5-fold increase in [55Fe]hemin binding sites, concomitant with the increase in [3H]PK11195 binding sites, as compared with those seen in antisense PBR/IBP cDNA-transfected cells. The binding of hemin to the transfected receptors exhibited a relatively high affinity with a Kd of 12 nM, and was inhibited by several benzodiazepine ligands, including PK11195, Ro 5-4864, diazepam and protoporphyrin IX. When mouse liver mitochondria were incubated with [55Fe]hemin, the binding to PBR had a Kd of 15 +/- 1.8 nM. The Bmax of [55Fe]hemin binding to the mitochondria was 6.88 +/- 0.76 pmol/mg of protein, a value consistent with that of [3H]PK11195 binding, with a lower affinity. Coproporphyrinogen III, a precursor porphyrin produced in the cytosol, is translocated into mitochondria, then is converted to protoporphyrinogen IX; this conversion decreased in the presence of benzodiazepine ligands. To examine whether this decrease was related to a decrease in the binding of coproporphyrinogen to the mitochondria, the effects of benzodiazepines on the binding of coproporphyrinogen were examined. As the binding was dose-dependently inhibited by PK11195, Ro 5-4864, and diazepam, porphyrins are likely to be endogenous ligands for PBR. We propose that PBR play a role in the intracellular transport of porphyrins and heme.

Animals↗

PubMed1995

Ionotropic mechanisms involved in postsynaptic inhibition by the endothelins of ganglionic transmission in dog cardiac sympathetic ganglia.

We investigated the effects of endothelin-1 and endothelin-3 (ET-1, ET-3) on the ganglionic transmission of cardiac sympathetic ganglia in vivo by the direct administration of agents to the ganglia through the right subclavian artery while monitoring the heart rate (HR) as an indicator of the ganglionic function in spinal dogs. The positive chronotropic responses to dimethylphenylpiperazinium (DMPP) and McN-A-343 administered to the ganglia were similarly inhibited by ET-1 (0.05-0.2 microg) and ET-3 (0.5-2 microg), but ET-1 was approximately 10 times more potent than ET-3. The inhibition induced by ETs was antagonized by endothelin ETA receptor antagonist BQ-123 (20 microg). This inhibition was unaffected by pretreatment with indomethacin given intravenously (i.v.), ruling out the possible involvement of endogenous prostaglandins production. The voltage-sensitive Ca2+ channel antagonist nifedipine had no effect on inhibition. However, the inhibition was antagonized by pretreatment with the low conductance Ca2+-activated potassium channel antagonists, such as apamin (20 microg intraarterially, i.a.), scyllatoxin (10 mug i.a.) and D-tubocurarine (0.6 mg i.a.). On the other hand, the voltage-sensitive K+ channel antagonist 4-aminopyridine (4-AP), ATP-dependent K+ channel antagonist, glibenclamide, and high-conductance Ca2+-activated K+ channel antagonists iberiotoxin and charybdotoxin failed to affect the inhibition by ETs. The results suggest that ETs inhibit the nicotinic and muscarinic ganglionic transmission through the ETA receptor-operated low-conductance Ca2+-activated potassium channel at postganglionic sites.

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗

PubMed1994

Site-directed mutagenesis of human ferrochelatase: identification of histidine-263 as a binding site for metal ions.

In nature, ferrochelatase catalyzes the insertion of ferrous ion into the porphyrin macrocycle of protoporphyrin IX to exclude two protons to form protoheme IX: other porphyrin substrates, including mesoporphyrin IX may be used in vitro. Based on the deduced amino-acid sequences, one histidine residue (H263 of human enzyme) is conserved among all ferrochelatases cloned from human to bacterial cells, and three histidine residues (H157, H341 and H388 of human enzyme) are conserved among eukaryotic ferrochelatases; no cysteine residue is conserved. To attempt to clarify the binding site of ferrous ion, we converted four highly conserved histidine residues in human ferrochelatase to alanine, using site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli, and iron- and zinc-chelating activities were examined. Mutants H157A and H388A lost most of their activities and concomitantly the enzyme became susceptible to proteolytic degradation. Kinetic studies with the residual activities showed no significant change of Km values for metal ions or for mesoporphyrin IX. Mutation at H341 did not alter the enzyme activities. Iron- and zinc-chelating activities of mutant H263A were reduced to 30% and 21% of the activities of the wild type, respectively. Moreover, this mutation resulted in 18- and 3.4-fold increases in Km values toward ferrous and zinc ions, respectively, while the Km value for mesoporphyrin remained unchanged. These results indicate that the binding site for metal ions in ferrochelatase is distinct from that for the porphyrin, and suggest that histidine-263 contributes significantly to the binding of metal ions. Maintenance of the structure of the protein molecule may involve functions related to histidine-157 and -388.

Alanine↗

PubMed1994

Overexpression in Escherichia coli, and one-step purification of the human recombinant ferrochelatase.

Ferrochelatase (EC 4.99.1.1), a mitochondrial inner membrane-bound protein, is the terminal enzyme of heme biosynthesis. The cDNA encoding the human mature ferrochelatase was placed under transcriptional control of T7 RNA polymerase in an Escherichia coli expression system. The bacteria produced large amounts of 42 kDa protein which reacted with anti-ferrochelatase antibodies. Expressed ferrochelatase exhibited iron- and zinc-chelating activities, and was found as a soluble protein. The recombinant enzyme has been purified to apparent homogeneity with a high yield, by one-step purification involving Blue-Sepharose chromatography. The purified enzyme which showed a molecular weight of about 40,000 by gel-filtration, functioned in a monomeric form. Km value for both mesoporphyrin IX and protoporphyrin IX with zinc was 12.5 microM. Km values for iron and zinc with mesoporphyrin IX were 6.7 microM and 11.8 microM, respectively. Zinc-chelating activity was markedly stimulated by palmitic acid, but iron-chelating activity remained unchanged. The above results were similar to those reported previously for mammalian ferrochelatase. The overexpression and the simple purification of a functional ferrochelatase exhibiting the same properties as natural enzyme will allow us to elucidate the mechanism of the enzyme reaction and structural changes of the mutated enzyme.

Amino Acid Sequence↗

PubMed1994

Induction of peripheral-type benzodiazepine receptors during differentiation of mouse erythroleukemia cells. A possible involvement of these receptors in heme biosynthesis.

To search for a possible role for peripheral-type benzodiazepine receptors (PBR) during erythroid differentiation, we cloned the PBR isoquinoline carboxamide-binding protein (PBR/IBP), an 18-kDa protein on PBR, from a mouse erythroleukemia (MEL) cell cDNA library. Sequence analysis revealed that PBR/IBP comprises 169 amino acid residues (M(r) 18,828), and has a high homology with PBR/IBP from other sources. The cDNA allows for the expression of active PBR/IBP, exhibiting a high affinity for isoquinoline carboxamide, [3H]PK11195, with Kd of 0.80 and 1.56 nM. RNA blot analysis revealed that treatment of MEL cells with dimethyl sulfoxide led to an increase in PBR/IBP mRNA (delta 1.0 kilobases) for up to 72 h, with a concomitant induction of mRNAs for heme biosynthetic enzymes, coproporphyrinogen oxidase and ferrochelatase. The induction of PBR/IBP mRNA was also observed in MEL cells induced with diazepam. The binding activity of [3H]PK11195 in MEL cells showed a high affinity with Kd of 0.69-2.13 nM, and increased during erythroid differentiation. The order of potency of different ligands to compete against [3H]PK11195 binding in induced MEL cells was PK11195 > protoporphyrin IX > diazepam > coproporphyrinogen III > coproporphyrin III > estazolam. In contrast to the induction of PBR/IBP in induced MEL cells, the voltage-dependent anion channel (mitochondrial porin) associated with PBR remained unchanged. These results suggest that PBR/IBP on PBR may be involved in porphyrin transport and may even be a critical factor in erythroid-specific induction of heme biosynthesis.

Amino Acid Sequence↗