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Biomedical subjects

K Igarashi

Publications and source records attributed to K Igarashi.

At least 55 records · Page 3Linked to original sources
PubMed2001

The promoter of mouse transcription repressor bach1 is regulated by Sp1 and trans-activated by Bach1.

The Maf recognition element (MARE) is regulated by both activators and repressors. Bach1 and Bach2 repress MARE-dependent transcription by forming heterodimers with Maf-related oncoproteins. In order to gain an understanding of the regulation of bach1 gene expression, we analyzed the structure of the mouse bach1 gene. Comparison of the exon-intron structure of the bach1 gene with those of other NF-E2-related genes indicated that bach1 and bach2 constitute an evolutionarily distinct subfamily among bZip factors. The bach1 promoter region contains two GC boxes that are important for its basal activity and are bound by Sp1 in K562 cell extracts. In addition, we found an evolutionarily conserved MARE-like element located downstream of the transcription initiation site. Deletion of this element resulted in a higher promoter activity in K562 cells. Bach1 trans-activated its own promoter depending on the presence of the MARE-like element in co-transfection assays. However, Bach1 did not bind to the MARE-like element in electrophoretic mobility shift assays (EMSA). These results suggest that Bach1 activates its own promoter indirectly by inhibiting the putative repressor. Such a positive feedback regulation by the repressor Bach1 may play an important role in maintaining the expression of Bach1 while consolidating repression of other genes with MARE.

Animals↗
PubMed2001

Novel alpha-amylase that is highly resistant to chelating reagents and chemical oxidants from the alkaliphilic Bacillus isolate KSM-K38.

A novel alpha-amylase (AmyK38) was found in cultures of an alkaliphilic Bacillus isolate designated KSM-K38. Based on the morphological and physiological characteristics and phylogenetic position as determined by 16S ribosomal DNA gene sequencing and DNA-DNA reassociation analysis, it was suggested that the isolate was a new species of the genus Bacillus. The enzyme had an optimal pH of 8.0 to 9.5 and displayed maximum catalytic activity at 55 to 60 degrees C. The apparent molecular mass was approximately 55 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was around pH 4.2. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltohexaose, maltoheptaose, and, in addition, maltose as major end products after completion of the reaction. The activity was not prevented at all by EDTA and EGTA at concentrations as high as 100 mM. Moreover, AmyK38 was highly resistant to chemical oxidation and maintained more than 80% of its original activity even after incubation for 1 h in the presence of excess H2O2 (1.8 M).

Bacillus↗
PubMed2001

Properties of a revertant of Escherichia coli viable in the presence of spermidine accumulation: increase in L-glycerol 3-phosphate.

Escherichia coli CAG2242 cells are deficient in the speG gene encoding spermidine acetyltransferase. When these cells were cultured in the presence of 0.5 to 4 mM spermidine, their viability was greatly decreased through the inhibition of protein synthesis by overaccumulation of spermidine. When the cells were cultured with a high concentration of spermidine (4 mM), a revertant strain was obtained. We found that a 55-kDa protein, glycerol kinase, was overexpressed in the revertant and that synthesis of a ribosome modulation factor and the RNA polymerase sigma(38) subunit, factors important for cell viability, was increased in the revertant. Levels of L-glycerol 3-phosphate also increased in the revertant. Transformation of glpFK, which encodes a glycerol diffusion facilitator (glpF) and glycerol kinase (glpK), to E. coli CAG2242 partially prevented the cell death caused by accumulation of spermidine. It was also found that L-glycerol 3-phosphate inhibited spermidine binding to ribosomes and attenuated the inhibition of protein synthesis caused by high concentrations of spermidine. These results indicate that L-glycerol 3-phosphate reduces the binding of excess amounts of spermidine to ribosomes so that protein synthesis is recovered.

Amino Acid Sequence↗
PubMed2001

Reconstitution of human beta-globin locus control region hypersensitive sites in the absence of chromatin assembly.

The human beta-globin genes are regulated by the locus control region (LCR), an element composed of multiple DNase I-hypersensitive sites (HS sites) located 5' to the genes. Various functional studies indicate that the LCR confers high-level, position-independent, and copy number-dependent expression to linked globin genes in transgenic mice. However, the structural basis for LCR function is unknown. Here we show that LCR HS sites can be reconstituted in an erythroid cell-specific manner on chromatin-assembled LCR templates in vitro. Surprisingly, HS2 and HS3 are also formed with erythroid proteins in the absence of chromatin assembly, indicating that sensitivity to nucleases is not simply a consequence of nucleosome reorganization. The generation of LCR HS sites in the absence of chromatin assembly leads to the formation of S1- and KMnO(4)-sensitive regions in HS2 and HS3. These sites are also sensitive to S1 nuclease in erythroid cells in vivo, suggesting a distorted DNA structure in the LCR core enhancer elements. Finally, we show that RNA polymerase II initiates transcription in the HS2 and HS3 core enhancer regions in vitro. Transcription in both HS2 and HS3 proceeds in a unidirectional manner. Taken together, the data suggest that erythroid proteins interact with the core enhancer elements, distort the DNA structure, and recruit polymerase II transcription complexes. These results further our understanding of the structural basis for LCR function and provide an explanation for why the LCR core regions are so extremely sensitive to nucleases in erythroid cells.

Animals↗
PubMed2001

Analysis of the metabolism of haloperidol and its neurotoxic pyridinium metabolite in patients with drug-induced parkinsonism.

The blood levels of the neurotrophic drug haloperidol (HP) and its pyridinium metabolite, HPP(+), have been analyzed by liquid chromatography/electrospray ionization-mass spectrometry in 10 schizophrenic patients treated with HP, without carbamazepine (HP, oral daily dose of 0.3-0.5 mg/kg body weight for more than 1 year, females, aged 41 +/- 8.5 years). There was a significant difference (t-test, d.f. = 8, p (t(0) = 7.2) <0.005) in the blood HPP(+) level between the 5 patients with (18.5 +/- 6.4 ng/ml) and the 5 without (6.3 +/- 2.4 ng/ml) severe side effects such as drug-induced parkinsonism (Extrapyramidal Symptom Rating Scale (ESRS) parkinsonism severity scores 2.8 +/- 1.5 and 1.8 +/- 1.1, respectively). Moreover, it is suggested that vitamin E may be effective for drug-induced parkinsonism through a change in the blood HPP(+) level. It is necessary to investigate the HPP(+) metabolism in psychiatric patients to avoid severe side effects such as drug-induced parkinsonism and cardiac functional disorders.

Adult↗
PubMed2001

Production and characterization of recombinant Phanerochaete chrysosporium cellobiose dehydrogenase in the methylotrophic yeast Pichia pastoris.

The hemoflavoenzyme cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. After 4 days of cultivation in the induction medium, the expression level reached 1800 U/L (79 mg/L) of CDH activity, which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P. chrysosporium. Analysis with SDS-PAGE and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with an approximate molecular mass of 100 kDa, which was identified as rCDH by Western blotting. The absorption spectrum of rCDH shows that the protein contains one flavin and one heme cofactor per protein molecule, as does wtCDH. The kinetic parameters for rCDH using cellobiose, ubiquinone, and cytochrome c, as well as the cellulose-binding properties of rCDH were nearly identical to those of wtCDH. From these results, we conclude that the rCDH produced by Pichia pastoris retains the catalytic and cellulose-binding properties of the wild-type enzyme, and that the Pichia expression system is well suited for high-level production of rCDH.

Carbohydrate Dehydrogenases↗
PubMed2001

Massive subchorionic hematoma (Breus' mole) complicated by intrauterine growth retardation.

We present here a case of massive subchorionic hematoma complicated by intrauterine growth retardation and oligohydramnios diagnosed at 22 weeks' gestation. The patient was managed with the following medications: (1) tocolysis with ritodrine infusion, (2) 10%maltose infusion therapy (1500mL/day), (3) antibiotic infusion (cefotaxim sodium, 2 g/dayx7) and (4) kampo therapy with Sairei-to until delivery. At 33 weeks and 0 days' gestation, a female baby weighing 1,342 g was delivered without complication by caesarean section. During surgery, an escape of about 500~600 g of dark brown blood with no clots was noted from the subchorionic space of the placenta. Examination of the placenta showed a large fibrosis with well-defined margins on the fetal surface.

Adult↗
PubMed2001

Microscopic polyangiitis associated with sinobronchial syndrome.

A woman with a long history of chronic bronchitis and chronic sinusitis, i.e., sinobronchial syndrome, was admitted with a fever. Radiologically, there were areas of longstanding consolidation in both lungs, with areas of active inflammation demonstrated by gallium-67 scintigraphy. Antineutrophil cytoplasmic antibody specific for myeloperoxidase was highly positive. Pulmonary hemorrhage and hematuria occurred 2 weeks after admission and responded to steroid therapy. However, the patient died of pneumonia. An autopsy revealed systemic necrotizing vasculitis affecting multiple organs, consistent with microscopic polyangiitis. The vasculitis might have been caused by the chronic inflammation in the lungs associated with sinobronchial syndrome.

Aged↗
PubMed2001

[Lung involvement in rapidly progressive glomerulonephritis].

The purpose of this study is to evaluate pulmonary involvement in rapidly progressive glomerulonephritis (RPGN). Of 71 patients in whom RPGN was diagnosed, 32 (45.1%) had pulmonary involvement: 12, alveolar hemorrhage (AH) (16.9%); 10, interstitial pneumonia (IP) (14.1%); four, chronic bronchial lesions (5.6%); two, consolidation (2.8%), and four, old inflammatory lesions (5.6%). Investigation of which organ of the twelve AH patients, the kidney or the lung, was the first to be involved, showed that the lung preceded in one patient, the kidney was first in three, and the lung and kidney occurred at the same time in the other eight. Of the ten IP patients, the lung preceded in eight, the kidney was ahead in two, and lung and kidney were simultaneous in one. Three patients had a history of pneumoconiosis, and two had acute progressive IP. Seventeen (45.9%) of 37 patients who tested positive for antineutrophil cytoplasmic autoantibodies (MPO-ANCA) had lung involvement, and three (30.0%) of the ten patients who tested negative for MPO-ANCA, PR 3-ANCA and anti-GBM antibody also had lung involvement.

Aged↗
PubMed2001

[A surgical treatment for ischemic heart disease associated with systemic lupus erythematodes: report of a case].

We reported a case of coronary artery bypass grafting (CABG) associated with systemic lupus erythematodes (SLE). A 45-year-old male who had been treated for SLE with prednisolone for 9 years was transferred to our department for a surgical treatment due to ischemic heart disease (IHD). We successfully performed CABG resolving various perioperative complications. The intensive care is indispensable in the case of IHD with SLE.

Anti-Inflammatory Agents↗
PubMed2001

[Susceptibilities of bacteria isolated from patients with lower respiratory infectious diseases to antibiotics (1999)].

From October 1999 to September 2000, we collected the specimen from 430 patients with lower respiratory tract infections in 17 institutions in Japan, and investigated the susceptibilities of isolated bacteria to various antibacterial agents and antibiotics and patients' characteristics. Of 515 strains that were isolated from specimen (mainly from sputum) and assumed to be bacteria causing in inflammation, 506 strains were investigated. The breakdown of the isolated bacteria were: Staphylococcus aureus 78, Streptococcus pneumoniae 101, Haemophilus influenzae 104, Pseudomonas aeruginosa (non-mucoid) 58, P. aeruginosa (mucoid) 11, Moraxella subgenus Branhamella catarrhalis 41, Klebsiella pneumoniae 18, etc. Of 78 S. aureus strains, those with 4 micrograms/ml or above of MIC of oxacillin (methicillin-resistant S. aureus: MRSA) occupied 57.7%. Vancomycin and arbekacin showed the most potent activities against MRSA without detection of ABK-resistant strain (MIC: 64 micrograms/ml) and decrease of VCM-sensitive strains those were found in 1998. The frequency of S. pneumoniae exhibiting low sensitivity to penicillin (penicillin-intermediate S. pneumoniae: PISP + penicillin-resistant S. pneumoniae: PRSP) decreased to 34.7% from 46.0% in 1998. The frequency of PRSP was 3.0%, being the least number after 1991. Carbapenems showed strong activities against S. pneumoniae. Especially, panipenem inhibited the growth of all 101 strains with MIC of 0.063 microgram/ml. Generally, all drugs showed strong activities against H. influenzae with MIC80s of 4 micrograms/ml or below. MICs of ofloxacin ranged between 0.063 microgram/ml and 4 micrograms/ml in 1998, however, those were 0.125 microgram/ml or below in all H. influenzae in 1999 showing the strongest activity. Tobramycin and ciprofloxacin showed strong activities against P. aeruginosa (both mucoid and non-mucoid) with MIC80s of 1 microgram/ml. Number of isolated P. aeruginosa (mucoid) was little as 11, however, the susceptibilities to all drugs were better than P. aeruginosa (non-mucoid). K. pneumoniae showed good susceptibilities to all drugs except for ampicillin with decreasing of low-sensitive strains compared to those detected in 1998. Also, all drugs generally showed strong activities against M. (B.) catarrhalis. MIC80s of all drugs were 2 micrograms/ml or below. The drug which showed the strongest activity was imipenem inhibiting all 41 strains with MIC of 0.063 microgram/ml. On the patients' characteristics, the number of patients aged 80 years or older who had been increased was decreased in 1999 in the distribution by age. The percentage of the elderly patients aged 70 years or older was 47.0%, which occupied almost a half number of the total patients as in the last year. As for the incidence by disease, bacterial pneumonia and chronic bronchitis were the highest. They were noted in 37.9% and 30.5% of the patients, respectively. In 1999, bronchial asthma was frequently observed as compared in recent years. It was noted in about 10% of the patients which is the same % as in bronchiectasis. We examined the number of strains from these patients with infections before and after administration of antibiotics. In patients with bacterial pneumonia, the number of isolated strains was almost the same between those before and after administration. However, in patients with chronic bronchitis, the number of strains remarkably decreased to less than the half of the total after administration of antibiotics in the last year, but it decreased to 2/3 of the total in 1999. On the administration of antibiotics and isolated bacteria by the day of administration, the bacteria which were isolated more before administration were H. influenzae in 28.4%, S. pneumoniae in 25.7%, M. (B.) catarrhalis in 12.0% and S. aureus in 10.6%. The frequency of S. aureus after administration over 15 days was almost the same as that before administration, but the frequency of P. aeruginosa (both mucoid and non-mucoid) was 36.8% which was higher than that before administration. The frequency of isolated S. pneumoniae was decreased after administration and none of them was isolated after completion of administration. However, that of H. influenzae was decreased to 7.1% after administration within 3 days, and many H. influenzae were isolated after completion of administration as 21.4%.

Adolescent↗
PubMed2000

Identification of the putrescine recognition site on polyamine transport protein PotE.

The PotE protein can catalyze both uptake and excretion of putrescine. The K(m) values of putrescine for uptake and excretion are 1.8 and 73 microm, respectively. Uptake of putrescine is dependent on the membrane potential, whereas excretion involves putrescine-ornithine antiporter activity. Amino acids involved in both activities were identified using mutated PotE proteins. It was found that Cys(62), Trp(201), Trp(292), and Tyr(425) were strongly involved in both activities, and that Tyr(92), Cys(210), Cys(285), and Cys(286) were moderately involved in the activities. Mutations of Tyr(78), Trp(90), and Trp(422) mainly affected uptake activity, and the K(m) values for putrescine uptake by these PotE mutants increased greatly, indicating that these amino acids are involved in the high affinity uptake of putrescine by PotE. Mutations of Lys(301) and Tyr(308) mainly affected excretion activity (putrescine-ornithine antiporter activity), and excretion by these mutants was not stimulated by ornithine, indicating that these amino acids are involved in the recognition of ornithine. It was found that the putrescine and ornithine recognition site on PotE is located at the cytoplasmic surface and the vestibule of the pore consisting of 12 transmembrane segments. Based on the results of competition experiments with various putrescine analogues and the disulfide cross-linking of PotE between cytoplasmic loops and the COOH terminus, a model of the putrescine recognition site on PotE consisting of the identified amino acids is presented.

Amino Acid Sequence↗
PubMed2000

Alpha-tocopherol transfer protein is important for the normal development of placental labyrinthine trophoblasts in mice.

Alpha-tocopherol transfer protein (alpha-TTP), a cytosolic protein that specifically binds alpha-tocopherol, is known as a product of the causative gene in patients with ataxia that is associated with vitamin E deficiency. Targeted disruption of the alpha-TTP gene revealed that alpha-tocopherol concentration in the circulation was regulated by alpha-TTP expression levels. Male alpha-TTP(-/-) mice were fertile; however, placentas of pregnant alpha-TTP(-/-) females were severely impaired with marked reduction of labyrinthine trophoblasts, and the embryos died at mid-gestation even when fertilized eggs of alpha-TTP(+/+) mice were transferred into alpha-TTP(-/-) recipients. The use of excess alpha-tocopherol or a synthetic antioxidant (BO-653) dietary supplement by alpha-TTP(-/-) females prevented placental failure and allowed full-term pregnancies. In alpha-TTP(+/+) animals, alpha-TTP gene expression was observed in the uterus, and its level transiently increased after implantation (4.5 days postcoitum). Our results suggest that oxidative stress in the labyrinth region of the placenta is protected by vitamin E during development and that in addition to the hepatic alpha-TTP, which governs plasma alpha-tocopherol level, the uterine alpha-TTP may also play an important role in supplying this vitamin.

Animals↗
PubMed2000

Transcription factor BACH1 is recruited to the nucleus by its novel alternative spliced isoform.

The transcription factor Bach1 is a member of a novel family of broad complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) basic region leucine zipper factors. Bach1 forms a heterodimer with MafK, a member of the small Maf protein family (MafF, MafG, and MafK), which recognizes the NF-E2/Maf recognition element, a cis-regulatory motif containing a 12-O-tetradecanoylphorbol-13-acetate-responsive element. Here we describe the gene structure of human BACH1, including a newly identified promoter and an alternatively RNA-spliced truncated form of BACH1, designated BACH1t, abundantly transcribed in human testis. The alternate splicing originated from the usage of a novel exon located 5.6 kilobase pairs downstream of the exon encoding the leucine zipper domain, and produced a protein that contained the conserved BTB/POZ, Cap'n collar, and basic region domains, but lacked the leucine zipper domain essential for NF-E2/Maf recognition element binding. Subcellular localization studies using green fluorescent protein as a reporter showed that full-length BACH1 localized to the cytoplasm, whereas BACH1t accumulated in the nucleus. Interestingly, coexpression of BACH1 and BACH1t demonstrated interaction between the molecules and the induction of nuclear import of BACH1. These results suggested that BACH1t recruits BACH1 to the nucleus through BTB domain-mediated interaction.

Alternative Splicing↗
PubMed2000

Differentiation of Toxocara canis and T. cati eggs by light and scanning electron microscopy.

In this study, we investigated the morphological identification of Toxocara canis and T. cati eggs on the basis of light and scanning electron microscopic (SEM) observations. T. canis and T. cati eggs used in this study were recovered from the uteri of respective gravid female worms. Measurement of egg size was not helpful in the differentiation of these species, because approximately 90% of eggs measured were of similar size. Using SEM, we were able to differentiate T. canis eggs from T. cati eggs based on their respective characteristic surface structures. Both species have subspherical eggs with markedly pitted surfaces like those of a golf ball, but the surface pitting in T. canis is more coarse than that in T. cati. In this study, however, these differences were not absolute, as 16% of T. canis and 29% of T. cati eggs showed surface pitting that was uncharacteristic of their species. Of the 16% of T. canis eggs that could not be differentiated by surface structure, 3% had pitting resembling T. cati, and the remaining 13% showed intermediate type surface pitting. Similarly, 5% of T. cati eggs resembled T. canis, and 25% of these were of intermediate type. Light microscopic observation yielded results similar to those of SEM, indicating that light microscopy is also a useful tool for the identification of Toxocara eggs.

Animals↗
PubMed2000

Suppression of rat thromboxane synthase gene transcription by peroxisome proliferator-activated receptor gamma in macrophages via an interaction with NRF2.

We have studied the transcription regulation of the rat thromboxane synthase (TXS) gene by peroxisome proliferator-activated receptor gamma (PPARgamma) in macrophages. The transcription activity of a cloned 5'-flanking region (1.6 kilobases) of the rat TXS gene (5'FL-TXS) was examined by luciferase reporter gene assay. TXS mRNA expression and the transcription activity of 5'FL-TXS were inhibited by PPARgamma ligands, 15-deoxy-Delta(12,14)-prostaglandin J(2) (PGJ(2)), and the thiazolidinedione troglitazone (TRO) in a dose-dependent manner. Overexpression of PPARgamma also significantly suppressed transcription, and further addition of PGJ(2) or TRO augmented the suppression. Deletion analysis showed that the element responsible for the PPARgamma effect is located in a region containing the nuclear factor E2 (NF-E2)/AP-1 site (-98/-88), which was indicated to be the major promoter of the TXS gene. By electrophoretic mobility shift assay using the NF-E2/AP-1 site and nuclear extracts from macrophages, we observed a specific protein-DNA complex formation, which was inhibited by a specific antibody against the transcription factor NRF2 (NF-E2-related factor 2). Moreover, the complex was decreased with PGJ(2), TRO, or in vitro translated PPARgamma. The transcription suppression by PPARgamma was confirmed using this truncated NRF2-binding element (-98/-88) by the reporter gene assay. Finally, a direct interaction between PPARgamma and NRF2 was confirmed by glutathione S-transferase pull-down assay. In conclusion, the NRF2-binding site (-98/-88) is the major promoter of 5'FL-TXS which can be suppressed by activated PPARgamma via a protein-protein interaction with NRF2 in macrophages.

Animals↗
PubMed2000

Increase in cap- and IRES-dependent protein synthesis by overproduction of translation initiation factor eIF4G.

The role of eIF4G during the initiation of protein synthesis was studied using mouse mammary carcinoma FM3A cells and FM4G cells that overproduce an N-terminally truncated form of eIF4G, which lacks the binding site of poly(A)-binding protein. An increase in eIF4G was correlated with an increase in protein synthesis and RNA helicase activity. Translation of mRNAshaving both short and long 5'-untranslated regions (5'-UTR) increased significantly in FM4G cells compared to that in FM3A cells. Both full-length and N-terminally truncated eIF4G transfectants of NIH3T3 cells formed colonies in soft agar and increased the saturation density of cell growth, indicating that both eIF4Gs function similarly. We also found that an internal ribosome entry site (IRES) exists in the 5'-UTR of ornithinedecarboxylase mRNA and that IRES-dependent protein synthesis increased in FM4G cells. Our results indicate that an increase in eIF4G contributes to the formation of active eIF4F similarly to that caused by an increase in eIF4E, as well as to a stimulation of IRES-dependent protein synthesis.

3T3 Cells↗