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N Ishida

Publications and source records attributed to N Ishida.

At least 145 records · Page 8Linked to original sources
PubMed1997

Expression of the human UDP-galactose transporter in the Golgi membranes of murine Had-1 cells that lack the endogenous transporter.

In our previous study, we demonstrated that UDP-galactose transporter cDNAs (hUGT1 and hUGT2) were able to complement the genetic defect of murine Had-1 cells that were deficient in the UDP-galactose transporter, and that the microsomal vesicles isolated from Had-1-transformants, which were obtained through transfection with these cDNAs, had recovered the ability to uptake UDP-galactose [Ishida, N. et al. (1996) J. Biochem. 120, 1074-1078]. In this report, we describe the preparation of peptide antibodies that recognize the hUGT isozymes, and the detection of hUGT proteins expressed in the transformants. The occurrence of the endogenous hUGT1 protein in HeLa cells was also detected. Using the hUGT1-specific antibodies, the subcellular localization of hUGT1 in the Golgi membrane was demonstrated by immunofluorescence microscopy and subcellular fractionation. These studies led us to develop a simple procedure, based on Percoll density gradient centrifugation, for preparing functional Golgi vesicles from the hUGT1-transformed Had-1 cells, that will facilitate future biochemical analyses of the UDP-galactose transporter for the elucidation of its structure-function relationship.

Amino Acid Sequence↗
PubMed1997

Mutation analysis and expression of the mottled gene in the macular mouse model of Menkes disease.

The gene for Menkes disease, an X-linked disorder of copper transport, has recently been identified and shown to encode a copper-transporting P-type ATPase. The macular mutant mouse has been proposed as an animal model for Menkes disease. In the present study, we report the finding of a missense mutation in the mottled gene of the macular mouse. A single base change, T to C, at nucleotide position 4223, is predicted to result in an amino acid change from serine to proline at residue 1382 in the eighth transmembrane domain. This mutation differs from the 6-bp deletion we find in brindled cDNA. With validation of macular as an animal model of Menkes disease, we compared mottled gene expression in the intestine, kidney, and brain of macular and normal mice. In Northern analyses an 8.3-kb transcript was detected in the intestine, kidney, and brain of both normal and macular mice, with the level of transcript in macular approximately 80% that of normal. In situ hybridization studies revealed that the mottled gene was clearly expressed in intestinal epithelial cells, Paneth cells, and renal proximal tubular cells of both normal and macular mice. In normal brain, mottled gene expression was most intensely observed in the choroid plexus, in Ammon's born and the dentate gyrus in the hippocampus, in Purkinje cells, and the granular layer of the cerebellum. The intensity and localization of the signals in the brain of macular mice were similar to those of the controls. The distribution of expression of mottled is correlated with cells and tissues showing histopathology or abnormal copper sequestration in macular and other mutants.

Adenosine Triphosphatases↗
PubMed1997

Transposon tagging in rice.

To develop an efficient gene isolation method for rice we introduced the maize Ac/Ds system into rice. Extensive analysis of their behavior in rice for several generations indicated that Ac and Ds in the presence of Ac transposase gene actively transpose in rice. A wide spectrum of mutations affecting growth, morphogenesis, flowering time and disease resistance have been obtained in the population carrying Ac/Ds and some of them were genetically analyzed. Main efforts are currently being made to isolate genes responsible these mutations. In addition, a number of Ac/Ds were mapped on chromosomes and mapped elements will be used in the future for directed tagging of genes with known chromosomal positions.

Cloning, Molecular↗
PubMed1997

Inhibition of cholesterol biosynthesis by squalene epoxidase inhibitor avoids apoptotic cell death in L6 myoblasts.

The relationship between the inhibition of cholesterol biosynthesis and occurrence of myopathy was studied in L6 myoblasts using two lines of cholesterol biosynthesis inhibitors, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor (simvastatin) and squalene epoxidase inhibitors (TU-2078 and NB-598). All inhibitors completely inhibited the cholesterol synthesis in L6 myoblasts at doses of 1 and 3 microM. Simvastatin (3 microM) inhibited the fusion reaction of L6 myoblasts followed by the severe cellular damage. The myoblasts also had failed actin fiber formation and creatinine phosphokinase (CPK) production. Additionally, this agent also caused apoptotic cell death in differentiated L6 muscle fiber, indicating that skeletal myopathy by HMG-CoA reductase inhibitors seems to occur not only in differentiating immature myoblasts but also in matured skeletal myotubes. In contrast, TU-2078 and NB-598 had no effect on the fusion reaction of differentiating myoblasts or on the cellular viability of muscle fiber at 3 microM, enough to completely inhibit cholesterol biosynthesis. It is conceivable that the mevalonate depletion and subsequent failure of ras farnesylation induced by simvastatin might cause the defects in differentiation and maintenance of the muscle fiber. Squalene epoxidase inhibitors did not show this adverse effect presumably because of the enzyme inhibition downstream of farnesyl synthesis. The present findings suggest the safe use of squalene epoxidase inhibitors in lipid-lowering therapy.

Actins↗
PubMed1996

c-fos CRE-binding activity of CREB/ATF family in the SCN is regulated by light but not a circadian clock.

The DNA-binding activity of cAMP-responsive element binding protein (CREB) in the suprachiasmatic nucleus (SCN) was examined with Ca or cAMP-responsive element (Ca/CRE) in upstream sequence of c-fos gene using electro-mobility shift assay (EMSA). By using supershift assay, Ca/CRE-binding activity in the SCN was shown to contain not only CREB but also activating transcription factor-1 (ATF-1). Furthermore, photic-induction of CREB binding activity to Ca/CRE in the SCN occurred at all circadian times tested, indicating that CREB DNA-binding in the SCN was not gated by the endogenous clock. These results implicated the role of CREB/ATF family in photic neuronal signaling in the SCN.

Activating Transcription Factor 1↗
PubMed1996

Sequence and neuronal expression of mouse endothelin-1 cDNA.

We have isolated and sequenced a cDNA that encodes mouse endothelin-1 (ET-1). The putative protein contains 202 amino acids corresponds to the prepro-form of ET-1. Twenty-one amino acids sequence of the putative mature ET-1 was identical with that of rat, porcine, bovine, and human. In situ hybridization histochemistry indicate that ET-1 mRNA was expressed in several hypothalamic nuclei including the suprachiasmatic nucleus (SCN) in rodent brain.

Amino Acid Sequence↗
PubMed1996

Expression and characterization of lacrimal gland water channels in Xenopus oocytes.

Lacrimal glands transport fluid for secretion as tears. To examine whether the expression of the aquaporin family of water channels is essential for water transport in lacrimal glands, we determined the water permeability in Xenopus oocytes injected with rat lacrimal gland poly(A)+ RNA. In oocytes injected with poly(A)+ RNA, osmotic water permeability was 4-fold higher than that observed in vehicle-injected controls. The enhanced water permeability was inhibited by 65% by coinjection of poly(A)+ RNA with antisense aquaporin-5 but not antisense aquaporin-1 oligonucleotide. To detect aquaporin mRNA in rat lacrimal glands, we performed reverse transcription-polymerase chain reaction analysis. PCR products were detected using specific aquaporin-5 primers. Our results strongly suggest that rat lacrimal glands express aquaporin-5 water channels for lacrimation.

Animals↗
PubMed1996

Mice lacking p27(Kip1) display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors.

SUMMARY: Mice lacking p27(Kip1) have been created by gene targeting in embryonic stem cells. These mice are larger than the control animals, with thymus, pituitary, and adrenal glands and gonadal organs exhibiting striking enlargement. CDK2 activity is elevated about 10-fold in p27(-/-) thymocytes. Development of ovarian follicles seems to be impaired, resulting in female sterility. Similar to mice with the Rb mutation, the p27(-/-) mice often develop pituitary tumors spontaneously. The retinas of the mutant mice show a disturbed organization of the normal cellular layer pattern. These findings indicate that p27(Kip1) acts to regulate the growth of a variety of cells. Unexpectedly, the cell cycle arrest mediated by TGFbeta, rapamycin, or contact inhibition remained intact in p27(-/-) cells, suggesting that p27(Kip1) is not required in these pathways.

Animals↗
PubMed1996

Overexpression of MP41 gene in a transformed endothelial cell line correlates with the increased fibronectin expression and a decreased incidence of tumorigenicity.

mp41 gene, which was originally identified as a gene regulated by the circadian clock, showed some similarity with yeast cell cycle regulator, CDC25, and its mRNA expression is restricted mainly to non-dividing cell. To elucidate the growth regulatory of mp41, mp41 cDNA was transfected into a transformed human endothelial cell line. All stable mp41 transformants showed increased levels of fibronectin expression and reduced migratory activity in vitro. Although the in vitro growth rate of mp41 transformants and their ability to grow in soft agar were not significantly altered, their tumor formation was suppressed significantly in nude mice. The results imply that mp41 gene overexpression altered the tumorigenicity associated with fibronectin elevation in stable transformation of the transformed endothelial cell line.

Animals↗
PubMed1996

Relationship of human liver dihydrodiol dehydrogenases to hepatic bile-acid-binding protein and an oxidoreductase of human colon cells.

We previously isolated three monomeric dihydrodiol dehydrogenases, DD1, DD2 and DD4, from human liver, and cloned a cDNA (C9) thought to encode DD2, which is identical with those for human bile-acid-binding protein and an oxidoreductase of human colon carcinoma HT29 cells. In the present study we have provided evidence that the C9 cDNA clone encodes DD1, not DD2. A recombinant enzyme expressed from the cDNA in a bacterial system was purified, and its catalytic properties, bile-acid-binding ability and primary sequence were compared with those of the hepatic dihydrodiol dehydrogenases. The results show that DD1 encoded by C9 possesses prostaglandin F synthase activity but low affinity for lithocholic acid, whereas DD2, showing differences of six amino acid residues from the DD1 sequence, exhibited high-affinity binding for the bile acid. Refined relationship between dihydrodiol dehydrogenases and their related proteins of human tissues is proposed.

Amino Acid Sequence↗
PubMed1996

Potent and long-lasting anticonvulsant effects of 1-naphthylacetyl spermine, an analogue of Joro spider toxin, against amygdaloid kindled seizures in rats.

The anticonvulsant effect of 1-naphthylacetyl spermine (1-NA-Spm), an analogue of Joro spider toxin, against amygdaloid kindled seizures was studied in rats. 1-NA-Spm (10, 20 and 40 micrograms/rat) dose-dependently improved kindled seizures and shortened the afterdischarge duration 30 min after the administration. The anticonvulsant effect was observed even one day after the drug, and then gradually disappeared within 4 days. The present findings demonstrate that 1-NA-Spm acts as a potent and long-acting anticonvulsant against amygdaloid kindled seizures, and also suggest, together with the previous findings, that the calcium-permeable AMPA receptors, which are selectively antagonized by 1-NA-Spm, play a critical role in the seizure generation mechanism of amygdaloid kindling.

Amygdala↗
PubMed1996

Circadian expression of the carbonic anhydrase gene, Cah1, in Chlamydomonas reinhardtii.

We have investigated whether the expression of carbonic anhydrase genes (Cah1 and Cah2) is regulated by a circadian clock in Chlamydomonas. When cells were grown in ordinary air under 12 h light/12 h dark (LD) cycles, the levels of the Cah2 mRNA hardly altered during the cycles, while the Cah1 mRNA showed a strong diurnal rhythm. The rhythm of about 24 h continued at least 3 days even under continuous light. Temperature compensation of the rhythm was demonstrated, using cultures maintained at 16, 22, and 28 degrees C. These results indicate that the abundance of the Cah1 transcript is controlled by a circadian clock.

Animals↗
PubMed1996

Expression of c-fos mRNA after audiogenic seizure in adult rats with neonatal hypothyroidism.

In situ hybridization histochemistry was used to evaluate the expression of the immediate-early gene c-fos following the induction of audiogenic seizures in adult rats with transient neonatal hypothyroidism. The rats treated with 0.02% propylthiouracil (PTU) through mother's milk during days 0-19 after delivery showed a high incidence of seizures to auditory stimulation at the age of 4 months. The significant induction of c-fos mRNA by audiogenic seizures is prominent in several brain areas including central gray, peripeduncular nucleus, inferior colliculus, septal nucleus, bed nucleus of stria terminalis, and dorsomedial hypothalamus. However, the expression of c-fos mRNA was comparable in neocortex, dorsal hippocampus and medial geniculate body between control rats and PTU-treated, seizure-induced rats. These results confirm the previous report on the c-fos expression following audiogenic seizure sensitized during development by a loud noise [20]. The present results indicate that the neonatal PTU treatment may provide a useful tool for studying the mechanism underlying the seizure susceptibility and development after maturation.

Acoustic Stimulation↗
PubMed1996

Enzyme-linked immunosorbent assay of neocarzinostatin chromophore (NCS-chr) by use of a monoclonal antibody against NCS-chr analog.

A monoclonal antibody against NCS-chr was prepared and characterized. Because of the instability of NCS-chr, chemically synthesized stable analog compound, termed PS, was used as a hapten of immunogen. The obtained antibody, termed APS, reacted with NCS-chr, but neither with NCS, NCS-polystyrene-maleic acid conjugate (SMANCS), nor UV-irradiated NCS-chr. Epitope analysis using the compounds that have a structure similar to PS showed that APS recognized the total structure, particularly cyclopenten moiety, of PS. These results suggest that APS recognizes the enediyne structure of NCS-chr. Next, the inhibition enzyme-linked immunosorbent assay (ELISA) for determination of NCS-chr was established. The standard curve showed that the microgram order of NCS-chr were accurately measurable by the established ELISA. Furthermore, it was revealed that the established ELISA was more sensitive than the antibiotic activity determination, termed Bio-assay. The established ELISA will be useful as a quantitative method of NCS-chr.

Antibiotics, Antineoplastic↗
PubMed1996

Human UDP-galactose translocator: molecular cloning of a complementary DNA that complements the genetic defect of a mutant cell line deficient in UDP-galactose translocator.

We have cloned a cDNA that codes for a putative human UDP-galactose translocator (UGT) protein. The cDNA contained an open reading frame of 1,179 base pairs encoding a novel protein of 393 amino acids. Introduction of the open reading frame sequence into a UGT-deficient mouse cell line, Had-1, complemented the genetic defect of the mutant, namely the inability to transport UDP-galactose from the cytosol to the Golgi lumen, as judged from the lectin-sensitivity spectrum of the transformant. To our knowledge, this is the first mammalian nucleotide-sugar translocator whose cDNA sequence has been described.

Amino Acid Sequence↗
PubMed1996

Molecular cloning and characterization of a novel isoform of the human UDP-galactose transporter, and of related complementary DNAs belonging to the nucleotide-sugar transporter gene family.

We described recently the molecular cloning of human UDP-galactose transporter 1 (hUGT1) [Miura, N. et al. (1996) J. Biochem. 120, 236-241]. Now we have characterized its isoform, hUGT2, that is most likely generated through the alternative splicing of a transcript derived from the UGT genomic gene, that also codes for hUGT1. Introduction of the open reading frame sequence of hUGT2 into a mouse cell line, Had-1, that lacks the UDP-galactose transporter, complemented the genetic defect of the mutant, as judged from the lectin-sensitivity spectra of the transformants and the nucleotide-sugar transporting activity of microsomal vesicles isolated from them. UGT-related genes were found through a BLAST search of dbEST based on their significant similarity with hUGT genes. We report here cDNA clones belonging to two subfamilies of the nucleotide-sugar transporter gene family. One is the human CMP-sialic acid transporter gene, and the other is a group of homologous genes with an undefined function that are distributed in man, mouse, and rat, and show significant similarity to the yeast UDP-N-acetylglucosamine transporter.

Amino Acid Sequence↗
PubMed1996

Effect of beraprost, a stable prostacyclin analogue, on red blood cell deformability impairment in the presence of hypercholesterolemia in rabbits.

We evaluated the effects of orally administered beraprost, a stable prostacyclin analogue, on the rheological behavior of red blood cells (RBC) in the presence of hypercholesterolemia. Rabbits fed a cholesterolrich diet were administered various doses of beraprost or pravastatin. We evaluated rheological behavior of RBC by assessing RBC deformability, using a positive-pressure filtration method. The maximum pressure generated by passing a suspension of RBC through a membrane filter was used as an index of RBC deformability. After animals were fed cholesterol for 16 weeks, the maximum pressure increased significantly from 172 +/- 15 mm Hg at baseline to 261 +/- 18 mm Hg (p < 0.01, n = 24). The reduction in RBC deformability associated with hypercholesterolemia improved dose dependently during 1-h incubation with various doses of beraprost. In ex vivo study, beraprost markedly restored RBC deformability 3 h after its oral administration to 218 +/- 17 mm Hg (n = 9) at a low dose and to 215 +/- 20 mm Hg (n = 9) at a high dose. The effect persisted for at least 2 h. Pravastatin failed to reduce the increased maximum pressure. Findings suggest that beraprost treatment may improve the microcirculation by restoring RBC deformability in the presence of hypercholesterolemia.

Animals↗