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Biomedical subjects

N Shimizu

Publications and source records attributed to N Shimizu.

At least 343 records · Page 19Linked to original sources
PubMed1998

Human retina-specific amine oxidase: genomic structure of the gene (AOC2), alternatively spliced variant, and mRNA expression in retina.

Previously, we reported the isolation of cDNA for human retina-specific amine oxidase (RAO) and the expression of RAO exclusively in retina. Bacterial artificial chromosome clones containing the human RAO gene (AOC2) were mapped to human chromosome 17q21 (Imamura et al., 1997, Genomics 40: 277-283). Here, we report the complete genomic structure of the RAO gene, including 5' flanking sequence, and mRNA expression in retina. The human RAO gene spans 6 kb and is composed of four exons corresponding to the amino acid sequence 1-530, 530-598, 598-641, and 642-729 separated by three introns of 3000, 310, and 351 bp. Screening of a human retina cDNA library revealed the existence of an alternatively spliced cDNA variant with an additional 81 bp at the end of exon 2. The sizes of exons and the locations of exon/intron boundaries in the human RAO gene showed remarkable similarity to those of the human kidney diamine oxidase gene (AOC1). In situ hybridization revealed that mRNA coding for RAO is expressed preferentially in the ganglion cell layer of the mouse retina. We designed four sets of PCR primers to amplify four exons, which will be valuable for analyzing mutations in patients with ocular diseases affecting the retinal ganglion cell layer.

Alternative Splicing↗
PubMed1998

Mouse myocilin (Myoc) gene expression in ocular tissues.

Human myocilin is identical to TIGR (trabecular meshwork inducible glucocorticoid response) which is responsible for the pathogenesis of juvenile-onset primary open angle glaucoma (GLCIA). We have isolated cDNA for mouse myocilin (Myoc) and investigated mouse myocilin gene expression in ocular tissues with in situ RNA hybridization. Hybridization signals were observed in the iris, ciliary body, trabecular meshwork, sclera, and retina in the mouse eye. The marked signals were seen in trabecular meshwork cells and the anterior portion of sclera. These findings suggest that myocilin mutation could affect the capacity of aqueous outflow and cause elevation of the intraocular pressure which is involved in the pathogenesis of glaucoma.

Amino Acid Sequence↗
PubMed1998

An efficient system for site-directed mutagenesis to make various mutants of the env gene of human immunodeficiency virus type 1.

We developed an efficient system of site-directed mutagenesis for the envelope (env) gene of human immunodeficiency virus type 1 (HIV-1). To make a template plasmid for mutagenesis, pS+B/MluI, two independent selection markers, i.e. a unique restriction site, MluI, and an in-frame termination codon, were introduced into the region encoding the V3 domain of the env gene of an HIV-1 strain, NL4-3, which had been cloned in the pUC118 plasmid. When the env gene of the pS+B/MluI plasmid was mutated successfully using mutagenic primers such as synthetic oligonucleotides or PCR-amplified DNA fragments longer than 1.5 kbp, the plasmids became resistant to digestion with MluI and competent env genes were formed by suppression of the in-frame termination. Various site-directed mutants of the env gene of HIV-1 were accurately constructed in a short time even in the absence of proper restriction sites by this system. The system of site-directed mutagenesis we reported here will be a useful method to analyze the functions of variable genes like the env gene of HIV-1 precisely and rapidly.

Amino Acid Sequence↗
PubMed1998

Novel nuclear localization signal between the two DNA-binding zinc fingers in the human vitamin D receptor.

The human vitamin D receptor (hVDR) possesses a unique array of five basic amino acids positioned between the two DNA-binding zinc fingers that is similar to well-characterized nuclear localization sequences in other proteins. When residues within this region are mutated to nonbasic amino acids, or when this domain is deleted, the receptor is still well expressed, but it no longer associates with the vitamin D-responsive element in DNA, in vitro, and hVDR-mediated transcriptional activation is abolished in transfected cells. Concomitantly, the mutated hVDRs exhibit a significant shift in hVDR cellular distribution favoring cytoplasmic over nuclear retention as assessed by subcellular fractionation and immunoblotting. Independent immunocytochemical studies employing a VDR-specific monoclonal antibody demonstrate that mutation or deletion of this basic domain dramatically attenuates hVDR nuclear localization in transfected COS-7 cells. Although wild-type hVDR is partitioned predominantly to the nucleus in the absence of the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone, treatment with ligand further enhances nuclear translocation, as it does to some degree in receptors with the basic region altered. The role of 1,25(OH)2D3 may be to facilitate hVDR heterodimerization with retinoid X receptors, stimulating subsequent DNA binding and ultimately enhancing nuclear retention. Taken together, these data reveal that the region of hVDR between Arg-49 and Lys-55 contains a novel constitutive nuclear localization signal, RRSMKRK.

Amino Acid Sequence↗
PubMed1998

Survival by Mac-1-mediated adherence and anoikis in phorbol ester-treated HL-60 cells.

During the exposure of human myelocytic leukemia HL-60 cells to phorbol diester, nonadherent cells die by apoptosis, but adherent cells survive and growth-arrest at G1 phase of the cell cycle. Here we have shown that the adherent cells rapidly died by apoptosis after forced detachment (anoikis), indicating that phorbol diester induced apoptosis by default. Dimethylsphingosine induced apoptosis in the adherent cells, and sphingosine-1-phosphate rescued the detached cells from apoptosis. Sphingosine kinase activity in adherent cells was higher than that in nonadherent cells and was decreased by forced detachment. It is likely that the phorbol diester-induced apoptosis and the adhesion-mediated survival are modulated by sphingosine and sphingosine-1-phosphate, respectively. The adherent cells were reverted and reproliferated when allowed to spontaneously detach from plastic surfaces by removal of phorbol diester. This result suggests that after removal of phorbol diester, the commitment signal of apoptosis by default is lost faster than the survival signal by adherence.

Apoptosis↗
PubMed1998

A novel unusual DNA structure formed in an inverted repeat sequence.

A potential to form a non-cruciform unusual DNA structure was shown at the inverted repeat DNA sequence of a fish satellite DNA. The recombinant plasmid harboring a member of the EcoRI satellite family of Sillago japonica (Percoidei, Sillaginidae) was subjected to S1 nuclease treatment, and the cutting sites were mapped by primer extension assay. The S1 nuclease attacked the 3'-half of the inverted repeat but not the middle part of symmetry under various salt conditions, suggesting that this unusual DNA structure is different from the DNA cruciform and a conventional intramolecular triplex structure. In the presence of 200 mM potassium chloride, the typical DNA cruciform has extruded, suggesting that certain purine-purine-pyrimidine base triads are involved in the formation of this unusual DNA structure. These results support the occurrence of a novel unusual DNA structure formed in the inverted repeat sequence.

Animals↗
PubMed1998

Helicobacter pylori infection enhances N-methyl-N-nitrosourea-induced stomach carcinogenesis in the Mongolian gerbil.

No previous report has demonstrated the relationship between Helicobacter pylori (HP) infection and gastric carcinogenesis in an experimental animal model. A total of 170 male Mongolian gerbils (MGs) were divided into nine groups (18 < or = n < or = 20 for each group). MGs of four groups were inoculated with HP before or after continuous N-methyl-N-nitrosourea (MNU) administration via the drinking water. Both intestinal-type and diffuse-type adenocarcinomas, including signet ring-cell carcinomas, were found at 40 weeks after the study commenced, but only in the HP inoculation groups with MNU exposure and not in the MNU alone or HP inoculation alone control groups. The present findings demonstrate that HP infection increases the incidence of MNU-induced adenocarcinoma of the glandular stomach in MGs.

Adenocarcinoma↗
PubMed1998

Osmium isotopic evidence for ancient subcontinental lithospheric mantle beneath the kerguelen islands, southern indian ocean

Upper mantle xenoliths found in ocean island basalts are an important window through which the oceanic mantle lithosphere may be viewed directly. Osmium isotopic data on peridotite xenoliths from the Kerguelen Islands, an archipelago that is located on the northern Kerguelen Plateau in the southern Indian Ocean, demonstrate that pieces of mantle of diverse provenance are present beneath the Islands. In particular, peridotites with unradiogenic osmium and ancient rhenium-depletion ages (to 1.36 x 10(9) years old) may be pieces of the Gondwanaland subcontinental lithosphere that were incorporated into the Indian Ocean lithosphere as a result of the rifting process.

Journal Article↗
PubMed1998

High-fidelity digital hybridization screening.

We developed a highly efficient screening method for minimizing the hybridization of high-density replica (HDR) filters and for allowing simultaneous use of numerous oligonucleotide probes for STS markers. We designated this method "digital hybridization (DH) screening," in which a binary n-bit ID number is given to each probe, and a series of probe mixtures is prepared in an arranged combination. The matrix pattern between probe mixtures and hybridization signals determines the relation between a particular STS marker(s) and the corresponding DNA clone(s). Here, we describe a successful DH screening of over 15,000 human BAC clones with 126 STS marker probes with 7-bit ID numbers, which required only 8 sets of HDR filter hybridizations. Our results indicate that DH screening can be performed with more than 1000 STS marker probes with only 10-bit ID numbers. The DH screening method is convenient, economical, and of high fidelity, and thus it should facilitate the construction of sequence-ready DNA contigs for the human genome as well as for genomes of various species. The principle of DH screening has various applications to the biological sciences.

Animals↗
PubMed1998

Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism.

Parkinson's disease is a common neurodegenerative disease with complex clinical features. Autosomal recessive juvenile parkinsonism (AR-JP) maps to the long arm of chromosome 6 (6q25.2-q27) and is linked strongly to the markers D6S305 and D6S253; the former is deleted in one Japanese AR-JP patient. By positional cloning within this microdeletion, we have now isolated a complementary DNA done of 2,960 base pairs with a 1,395-base-pair open reading frame, encoding a protein of 465 amino acids with moderate similarity to ubiquitin at the amino terminus and a RING-finger motif at the carboxy terminus. The gene spans more than 500 kilobases and has 12 exons, five of which (exons 3-7) are deleted in the patient. Four other AR-JP patients from three unrelated families have a deletion affecting exon 4 alone. A 4.5-kilobase transcript that is expressed in many human tissues but is abundant in the brain, including the substantia nigra, is shorter in brain tissue from one of the groups of exon-4-deleted patients. Mutations in the newly identified gene appear to be responsible for the pathogenesis of AR-JP, and we have therefore named the protein product 'Parkin'.

Adult↗
PubMed1998

Involvement of Mac-1-mediated adherence and sphingosine 1-phosphate in survival of phorbol ester-treated U937 cells.

Phorbol esters exert a dual function in human leukemia cells, induction of differentiation and activation of integrin-mediated functions. Here we have shown that the plastic adherence of phorbol ester-treated U937 cells is mediated by expression of integrin Mac-1 (CD11b/CD18) on the cell surface and that these adherent cells exhibit anoikis (apoptosis when adherent cells are detached or adherence is inhibited). We used U937-derived clones overexpressing either antisense RNAs antisense to CD11b and CD18 mRNAs or mRNA from a truncated mutant CD11b gene. We have also shown that apoptosis in non-adherent cells or anoikis was mediated by sphingosine and that survival of adherent cells was achieved by a shift of the dynamic balance between sphingosine and sphingosine 1-phosphate toward the latter by adherence-activated sphingosine 1-kinase.

Apoptosis↗
PubMed1998

Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase.

Acentric, autonomously replicating extrachromosomal structures called double-minute chromosomes (DMs) frequently mediate oncogene amplification in human tumors. We show that DMs can be removed from the nucleus by a novel micronucleation mechanism that is initiated by budding of the nuclear membrane during S phase. DMs containing c-myc oncogenes in a colon cancer cell line localized to and replicated at the nuclear periphery. Replication inhibitors increased micronucleation; cell synchronization and bromodeoxyuridine-pulse labeling demonstrated de novo formation of buds and micronuclei during S phase. The frequencies of S-phase nuclear budding and micronucleation were increased dramatically in normal human cells by inactivating p53, suggesting that an S-phase function of p53 minimizes the probability of producing the broken chromosome fragments that induce budding and micronucleation. These data have implications for understanding the behavior of acentric DNA in interphase nuclei and for developing chemotherapeutic strategies based on this new mechanism for DM elimination.

Antimetabolites↗
PubMed1998

Role of nitric oxide in the hypersusceptibility to pentylenetetrazole-induced seizure in diazepam-withdrawn mice.

The decrease in the seizure threshold for pentylenetetrazole in diazepam-withdrawn mice was not significantly affected by L-arginine (50 and 100 microg/mouse, i.c.v.), which did have an antiseizure effect in chronically vehicle-treated mice. Sodium nitroprusside (25 and 50 microg/mouse, i.c.v.) increased the seizure threshold for pentylenetetrazole in both diazepam-withdrawn mice and chronically vehicle-treated mice. In addition, the antiseizure effect of L-arginine was blocked by the nitric oxide (NO) synthase inhibitor, N-nitro-L-arginine (NOARG) and the NO scavenger, hemoglobin, while the effect of sodium nitroprusside was inhibited by hemoglobin, but not by NOARG, indicating that the antiseizure effect of L-arginine, but not that of sodium nitroprusside, is mediated by NO production resulting from the activation of NO synthase. Therefore, a decrease in the NO production via NO synthase may be involved in the hypersusceptibility to pentylenetetrazole during diazepam withdrawal.

Animals↗
PubMed1998

Identification of the chemokine receptor TER1/CCR8 expressed in brain-derived cells and T cells as a new coreceptor for HIV-1 infection.

We have isolated HIV-1 variants that infect brain-derived CD4-positive cells, which are resistant to both macrophage (M)-tropic and T-cell line (T)-tropic HIV-1 strains. It is possible that this brain cell tropism of the HIV-1 variants is determined by the interaction of HIV-1 with a chemokine receptor (CKR) gene. We attempted to detect the expression of the CKR-like genes using degenerate PCR primers. The brain-derived cells expressed a CKR-like gene TER1/CCR8. Human CD4-expressing cells resistant to all HIV-1 strains became susceptible to brain-cell tropic HIV-1 variants after expression of TER1 in these cells, but these cells were still resistant to M-tropic strains or T-tropic IIIB strain. TER1 was expressed in brain-derived cells and human T-cells. These findings suggest that TER1/CCR8 functions as a co-receptor for HIV-1 infection for brain-derived cells as well as T cells.

Brain↗
PubMed1998

Antisense oligonucleotide of WAF1 gene prevents EGF-induced cell-cycle arrest in A431 cells.

A431 cells hyperproduce EGF receptors and possess inactive p53 proteins. It has been suggested that a cyclin-dependent kinase (CDK) inhibitor p21/WAF1 plays a crucial role in the EGF-induced cell-cycle arrest of A431 cells. Here, we investigated the role of WAF1 gene transcription in the EGF-induced cell-cycle arrest by transfecting the 18-mer antisense oligonucleotide which corresponds to the 5' region of WAF1 gene (AS/WAF1). When A431 cells were treated with EGF, a cascade of responses were observed, including immediate hyperphosphorylation of EGF receptor on tyrosine residues, accumulation of WAF1 mRNA and p21/WAF1 protein, dephosphorylation of RB protein which is a substrate of CDK-cyclin, and cell-cycle arrest. In the presence of AS/WAF1, EGF induced the tyrosine-phosphorylation of EGF receptor, but WAF1 mRNA was reduced to a half; accumulation of p21/WAF1 protein and its downstream responses were no longer observed; A431 cells grew continuously. Thus, the transfection of antisense efficiently prevented A431 cells from the EGF-induced arrest. These observations suggest that p21/WAF1 protein is a major effector molecule of the EGF-mediated cell-cycle arrest of A431 cells.

Cell Cycle↗
PubMed1998

Helicobacter pylori promotes development of pepsinogen-altered pyloric glands, a preneoplastic lesion of glandular stomach of BALB/c mice pretreated with N-methyl-N-nitrosourea.

H. pylori is thought to be a stomach carcinogen. Since no experimental model has hitherto been established to clarify the relationship between H. pylori and stomach carcinogenesis, the effects of infection with the bacteria on experimental carcinogenesis in the glandular stomach of mice were investigated. BALB/c mice were given salty diet or N-methyl-N-nitrosourea (MNU) and administered broth culture of H. pylori. The incidence of pepsinogen-altered pyloric glands, considered as precancerous lesions, was increased in the H. pylori inoculated group pre-treated with MNU. The findings provide the new experimental model demonstrating the relationship between stomach cancer and H. pylori.

Animals↗
PubMed1998

Nuclear targeted suppression of NF-kappa B activity by the novel quinone derivative E3330.

The activation of NF-kappa B consists of at least three steps: degradation of I kappa B alpha, translocation of NF-kappa B into the nucleus, ai post-translational modification of NF-kappa B (e.g., phosphorylation of p65). In the present study, we found that a novel quinone derivative E3330 selectively inhibited NF-kappa B-mediated gene expression without affecting any of these steps. E3330, when included in the culture medium, suppressed NF-kappa B DNA-binding activity in PMA-induced Jurkat cell nuclear extracts, suggesting that the inhibition by E3330 of NF-kappa B-mediated gene expression was due to its ability to suppress NF-kappa B DNA-binding activity. Fractionation of the nuclear extracts by column chromatography revealed that a nuclear factor enhanced NF-kappa B DNA-binding activity and that this enhancing activity was interrupted after treatment with E3330. Moreover, a major polypeptide with a molecular mass of 40 kDa was found to be in the highly purified fraction containing the NF-kappa B-enhancing activity and predominantly bind E3330. Taken together, these results suggest that the NF-kappa B activity, after dissociation from I kappa B, is enhanced by a nuclear factor that is active irrespective of PMA treatment, and the nuclear factor-mediated enhancement is selectively inhibited by E3330. Thus, we conclude that E3330 may belong to a novel class of anti-NF-kappa B drugs.

Benzoquinones↗