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Biomedical subjects

S Nishikawa

Publications and source records attributed to S Nishikawa.

At least 127 records · Page 7Linked to original sources
PubMed1999

Structural study of an RNA aptamer for a Tat protein complexed with ligands.

An RNA aptamer for an HIV Tat protein has been isolated by the in vitro SELEX method. The RNA aptamer binds to the Tat protein 50-100 times more strongly than native TAR RNA does. Here, we have investigated the structure of the RNA aptamer complexed with ligands, partial peptide fragments of the Tat protein or argininamide, by multidimensional 1H/13C/15N NMR. It is strongly suggested that two U:A:U base triples are formed in the RNA aptamer upon binding of ligands. Specific hydrogen bonds between arginine side chains of ligands and guanine bases located adjacent to the base triples are identified. On the basis of many intramolecular and intermolecular NOEs, a structural model of the complex has been constructed.

Base Sequence↗
PubMed1999

Importance of short pseudoknot base pairs between two single-stranded regions of HDV ribozyme.

Human hepatitis delta virus (HDV) ribozyme can catalyze self-cleavage reaction in the presence of Mg2+ ions, yielding products with 2',3'-cyclic phosphate and 5'-OH termini as do hammerhead and hairpin ribozymes. Recently, the tertiary structure of 3'-cleaved product of genomic HDV ribozyme was solved by X-ray crystallographic analysis. In this structure three single-stranded regions (SSrA, -B and -C) interacts intricately with hydrogen bonds between bases, phosphate oxygens and 2'-OHs to form nested double pseudoknot structure. Especially two Watson-Crick base pairs, 726G-710C and 727G-709C, between SSrA and SSrC, seems to be important for compact folding. To characterize the necessity of the two base pairs, we performed in vitro selection of active ribozymes using random RNA pool which mutated at 709, 710, 726 and 727. The result indicates that basically one G-C base pair is necessary for the activity.

Base Pairing↗
PubMed1999

Genetic and biochemical characterization of a 2-pyrone-4, 6-dicarboxylic acid hydrolase involved in the protocatechuate 4, 5-cleavage pathway of Sphingomonas paucimobilis SYK-6.

Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds with guaiacyl moieties, which are converted into protocatechuate by the actions of lignin degradation enzymes in this strain. Protocatechuate is a key metabolite in the SYK-6 degradation of lignin compounds with guaiacyl moieties, and it is thought that it degrades to pyruvate and oxaloacetate via the protocatechuate 4,5-cleavage pathway. In a 10.5-kb EcoRI fragment carrying the protocatechuate 4,5-dioxygenase gene (ligAB) (Y. Noda, S. Nishikawa, K. Shiozuka, H. Kadokura, H. Nakajima, K. Yoda, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki. J. Bacteriol. 172:2704-2709, 1990), we found the ligI gene encoding 2-pyrone-4, 6-dicarboxylic acid (PDC) hydrolase. PDC hydrolase is a member of this pathway and catalyzes the interconversion between PDC and 4-carboxy-2-hydroxymuconic acid (CHM). The ligI gene is thought to be transcribed divergently from ligAB and consists of an 879-bp open reading frame encoding a polypeptide with a molecular mass of 32,737 Da. The ligI gene product (LigI), expressed in Escherichia coli, was purified to near-homogeneity and was estimated to be a monomer (31.6 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9. The optimum pH for hydrolysis of PDC is 8.5, the optimum pH for synthesis of PDC is 6.0 to 7.5, and the Km values for PDC and CHM are 74 and 49 microM, respectively. LigI activity was inhibited by the addition of thiol reagents, suggesting that the cysteine residue is a catalytic site. LigI is more resistant to metal ion inhibition than the PDC hydrolases of Pseudomonas ochraceae (K. Maruyama, J. Biochem. 93:557-565, 1983) and Comamonas testosteroni (P. J. Kersten, S. Dagley, J. W. Whittaker, D. M. Arciero, and J. D. Lipscomb, J. Bacteriol. 152:1154-1162, 1982). The insertional inactivation of the ligI gene in S. paucimobilis SYK-6 led to the complete loss of PDC hydrolase activity and to a growth defect on vanillic acid; it did not affect growth on syringic acid. These results indicate that the ligI gene is essential for the growth of SYK-6 on vanillic acid but is not responsible for the growth of SYK-6 on syringic acid.

Carboxylic Ester Hydrolases↗
PubMed1999

The yeast RER2 gene, identified by endoplasmic reticulum protein localization mutations, encodes cis-prenyltransferase, a key enzyme in dolichol synthesis.

As an approach to understand the molecular mechanisms of endoplasmic reticulum (ER) protein sorting, we have isolated yeast rer mutants that mislocalize a Sec12-Mfalpha1p fusion protein from the ER to later compartments of the secretory pathway (S. Nishikawa and A. Nakano, Proc. Natl. Acad. Sci. USA 90:8179-8183, 1993). The temperature-sensitive rer2 mutant mislocalizes different types of ER membrane proteins, suggesting that RER2 is involved in correct localization of ER proteins in general. The rer2 mutant shows several other characteristic phenotypes: slow growth, defects in N and O glycosylation, sensitivity to hygromycin B, and abnormal accumulation of membranes, including the ER and the Golgi membranes. RER2 and SRT1, a gene whose overexpression suppresses rer2, encode novel proteins similar to each other, and their double disruption is lethal. RER2 homologues are found not only in eukaryotes but also in many prokaryote species and thus constitute a large gene family which has been well conserved during evolution. Taking a hint from the phenotype of newly established mutants of the Rer2p homologue of Escherichia coli, we discovered that the rer2 mutant is deficient in the activity of cis-prenyltransferase, a key enzyme of dolichol synthesis. This and other lines of evidence let us conclude that members of the RER2 family of genes encode cis-prenyltransferase itself. The difference in phenotypes between the rer2 mutant and previously obtained glycosylation mutants suggests a novel, as-yet-unknown role of dolichol.

Alkyl and Aryl Transferases↗
PubMed1999

Apoptosis of dental pulp cells and their elimination by macrophages and MHC class II-expressing dendritic cells.

Apoptosis of dental pulp cells of rat incisors was investigated by the TUNEL method and electron microscopy. The results showed that a considerable amount of apoptosis occurred in the pulp, increasing in extent with incisal direction. OX6, ED1, and ED2 antibodies were used to detect macrophages and dendritic cells in combination with immunoelectron microscopy. Apoptotic fragments were eliminated mainly by MHC Class II-expressing cells, including dendritic cells positive for the OX6 antibody, and by MHC Class II-negative macrophages. Macrophages and dendritic cells positive for OX6, ED1, or ED2 increased from the apical to incisal direction of the incisor. These results indicate that apoptosis contributes to normal pulp formation and maintenance.

Animals↗
PubMed1999

Analysis of the retinal edema of full-thickness macular holes by scanning laser ophthalmoscopy and optical coherence tomography.

The purpose of this study was to investigate the relationship between the dark area illuminated by scanning laser ophthalmoscopy (SLO) and cystic spaces around macular holes as shown by optical coherence tomography (OCT). SLO allows for two dimensional retinal examination, using short wave length (514 nm, argon) which is useful for the vitreoretinal surface and inner retina; red helium-neon laser (633 nm), which is capable of imaging deeper tissues; and infrared diode laser (780 nm), for choroidal examination. OCT is analogous to ultrasound except that optical rather than acoustic reflectivity is measured. OCT can produce the cross-sectional view of retina. Using SLO (helium-neon laser) and OCT, we examined 8 eyes with full-thickness macular holes. Eight normal eyes served as controls. Cystic spaces were in proportion to dark areas with statistical significance as shown by correlation analysis. Evaluation of the full-thickness macular holes by using both SLO and OCT is highly useful.

Aged↗
PubMed1998

Use of Bombyx mori silk fibroin as a substratum for cultivation of animal cells.

The growth of animal cells on silk fibroin-coated plates was examined. The anchorage-dependent cells showed almost the same growth on both fibroin- and collagen-coated plates, and it was 30-50% higher than that on polystyrene plates coated with hydrophilic groups. On the other hand, the growth of the anchorage-independent hybridomas on the three different plates did not show a significant difference. The cells grown on the respective plates produced their products with the same efficiency despite the difference in the chemical properties of the plates. In this paper, it is demonstrated that silk fibroin can be used as the substratum for the culture of animal cells in place of collagen.

Animals↗
PubMed1998

Host resistance against Listeria monocytogenes is reciprocal during the course of infection in alymphoplastic aly mutant mice.

The aly is a unique spontaneous autosomal recessive mutation in mice that causes a systemic defect of lymph nodes and Peyer's patches. We investigated host resistance against Listeria monocytogenes infection in the mutant. The 50% lethal dose of L. monocytogenes in aly/aly mice was 10-fold higher than their heterozygotes, termed aly/+mice, or their wild-type C57BL/6 mice. The bacterial growth in the spleens and livers of aly/aly mice was more efficient early in infection, and their listericidal activity of peritoneal macrophages was higher than those of aly/+mice. In contrast, the complete elimination of bacteria from the spleens and livers of infected mice in the late stage of infection, in which a T-cell-dependent mechanism is required, was delayed in the aly/aly mice. Moreover, an acquired resistance against secondary infection with L. monocytogenes was markedly diminished in the aly/aly mice. The production of endogenous interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), which are critical in antilisterial resistance, was reduced in the aly/aly mice during the infection. The production of IFN-gamma, and interleukin-4 was also diminished in the spleen cell cultures of aly/aly mice when stimulated with heat-killed L. monocytogenes or the T-cell receptors were directly stimulated with anti-CD3-epsilon monoclonal antibody. These results suggest that acquired immunity against L. monocytogenes infection is attenuated in aly/aly mice, and that the insufficient production of IFN-gamma and TNF-alpha might be involved in the immunodeficiency.

Animals↗
PubMed1998

The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract.

Vascularization of organs generally occurs by remodelling of the preexisting vascular system during their differentiation and growth to enable them to perform their specific functions during development. The molecules required by early vascular systems, many of which are receptor tyrosine kinases and their ligands, have been defined by analysis of mutant mice. As most of these mice die during early gestation before many of their organs have developed, the molecules responsible for vascularization during organogenesis have not been identified. The cell-surface receptor CXCR4 is a seven-transmembrane-spanning, G-protein-coupled receptor for the CXC chemokine PBSF/SDF-1 (for pre-B-cell growth-stimulating factor/stromal-cell-derived factor), which is responsible for B-cell lymphopoiesis, bone-marrow myelopoiesis and cardiac ventricular septum formation. CXCR4 also functions as a co-receptor for T-cell-line tropic human immunodeficiency virus HIV-1. Here we report that CXCR4 is expressed in developing vascular endothelial cells, and that mice lacking CXCR4 or PBSF/SDF-1 have defective formation of the large vessels supplying the gastrointestinal tract. In addition, mice lacking CXCR4 die in utero and are defective in vascular development, haematopoiesis and cardiogenesis, like mice lacking PBSF/SDF-1, indicating that CXCR4 is a primary physiological receptor for PBSF/SDF-1. We conclude that PBSF/SDF-1 and CXCR4 define a new signalling system for organ vascularization.

Animals↗
PubMed1998

Murine cutaneous mastocytosis and epidermal melanocytosis induced by keratinocyte expression of transgenic stem cell factor.

The growth and differentiation of mast cells and melanocytes require stem cell factor (SCF), the ligand for the kit receptor tyrosine kinase. SCF may exist as a membrane-bound or soluble molecule. Abnormalities of the SCF-kit signaling pathway, with increased local concentrations of soluble SCF, have been implicated in the pathogenesis of the human disease cutaneous mastocytosis, but have not yet been shown to play a causal role. To investigate both the potential of SCF to cause mastocytosis and its role in epidermal melanocyte homeostasis, we targeted the expression of SCF to epidermal keratinocytes in mice with two different transgenes controlled by the human keratin 14 promoter. The transgenes contained cDNAs that either produced SCF, which can exist in both membrane-bound and soluble forms, or SCF, which remains essentially membrane bound. Murine epidermal keratinocyte expression of membrane-bound/ soluble SCF reproduced the phenotype of human cutaneous mastocytosis, with dermal mast cell infiltrates and epidermal hyperpigmentation, and caused the maintenance of a population of melanocytes in the interadnexal epidermis, an area where melanocytes and melanin are found in human skin but where they are not typically found in murine skin. Expression of membrane-bound SCF alone resulted in epidermal melanocytosis and melanin production, but did not by itself cause mastocytosis. We conclude, first, that a phenotype matching that of human mastocytosis can be produced in mice by keratinocyte overproduction of soluble SCF, suggesting a potential cause of this disease. Second, we conclude that keratinocyte expression of membrane-bound SCF results in the postnatal maintenance of epidermal melanocytes in mice. Since the resulting animals have skin that more closely approximates human skin than do normal mice, their study may be more relevant to human melanocyte biology than the study of skin of normal mice.

Animals↗
PubMed1998

Multivalent DNA binding complex generated by small Maf and Bach1 as a possible biochemical basis for beta-globin locus control region complex.

The human beta-globin locus control region (LCR) is required to properly regulate chromatin domain opening, replication timing, and globin gene activation. The LCR contains multiple NF-E2 sites (Maf recognition elements, MAREs) that allow the binding of various basic leucine zipper (bZip) proteins like p45 NF-E2, Nrf1, Nrf2, Bach1, and Bach2, in some cases as obligate heterodimers with a small Maf protein. In addition to the bZip domain, the Bach proteins bear a BTB/POZ domain, which has been implicated in the regulation of chromatin structure. We show here that Bach1 is highly expressed in hematopoietic cells and constitutes one of the two MARE-binding activities in murine erythroleukemic (MEL) cells. We further demonstrate that Bach1/MafK heterodimers interact with each other through the BTB domain, generating a multimeric and multivalent DNA binding complex. These results strongly implicate Bach1/MafK heterodimer as an architectural transcription factor that mediates interactions among multiple MAREs. Such a factor could then provide a model for assembly of the theoretical beta-globin LCR "holocomplex. " Other BTB domain proteins have already been demonstrated to be involved in remodeling chromatin, and thus this class of proteins likely promote the formation of nucleoprotein complexes required to establish the architecture of regulatory domains.

Animals↗
PubMed1998

Reinvestigation of the functions of the hydrophobic segment of Jem1p, a yeast endoplasmic reticulum membrane protein mediating nuclear fusion.

Jem1p is a DnaJ-like protein of the yeast ER membrane, which is required for nuclear membrane fusion during mating. We have determined the position of translation initiation codon for the JEM1 gene. Translation of Jem1p starts from the second ATG codon of the previously assumed JEM1 open reading frame, leading to the synthesis of a precursor protein of 645 amino acids long. The translated Jem1p precursor contains an N-terminal hydrophobic sequence that functions as a signal sequence and is removed upon import into the ER lumen. Jem1p is peripherally associated with the ER membrane probably through protein-protein interactions.

Cell Compartmentation↗
PubMed1998

Selection of a RNA aptamer that binds to human activated protein C and inhibits its protease function.

A high-affinity RNA aptamer to human activated protein C (APC) was selected from a pool of random sequences using in vitro selection. Activated protein C, a trypsin-like serine protease plays an important role along with thrombin as a regulator in blood clotting cascade. After seven rounds of selection and amplification, a single predominant nucleic acid sequence APC-167, a 167-base oligonucleotide with a random sequence core of 120 bases, was obtained. The selected aptamer did not bind to thrombin or factor Xa and thus demonstrated specificity to APC. Furthermore, this aptamer was a non-competitive inhibitor to the cleavage reaction of a fluorogenic substrate catalyzed by APC. The inhibition constant (Ki) of APC-167 was 83 nM. The 99-base oligonucleotide (APC-99) derived from APC-167 by deleting both primer binding sites, was also found to inhibit APC strongly (Ki = 137 nM). Two stem-loop structures and at least one G x U wobble base pair in the stem were elucidated as important structural motifs for binding.

Base Composition↗
PubMed1998

Neovascularization of the RPE: temporal differences in mice with rod photoreceptor gene defects.

Neovascularization (NV) of the retinal pigment epitheium (RPE) by retinal capillaries following degeneration and loss of photoreceptor cells is a widely recognized phenomenon in rodents. NV of the RPE usually occurs several weeks to months after the loss of photoreceptor cells. We have observed that NV of the RPE occurs much earlier in a line of P23H mutant rhodopsin transgenic mice than in most other mice and rats that have been previously examined. To compare the temporal course of RPE NV in P23H mice with that of two other retinal degeneration mutants with the same time course of photoreceptor cell loss, we have quantified the number of retinal capillaries in the RPE of P23H and Q344ter mutant rhodopsin transgenic mice and retinal degeneration (rd/rd) mice at ages ranging from postnatal day (P) 20-400. Retinal capillary profiles located within the RPE were already present as early as P20 in the P23H retinas, and although these usually were located where most photoreceptor nuclei were missing, they occasionally were found where 1-2 rows of photoreceptor nuclei were still present. The maximal incidence was found in P23H retinas at P100. By contrast, NV of the RPE in rd/rd and Q344ter mice occurred much later. In rd/rd, a significant number of capillary profiles was not seen in the RPE until about P130, and not until about P180 in Q344ter. Both showed maximal incidence at about P240. In all three mutants, an apparent regression of the capillaries occurred following the peak, with that in the P23H mice preceeding the other two mutants. The findings suggest that the P23H mutant rhodopsin transgenic mouse may be a useful model for studying the regulation of NV in the outer retina.

Aging↗
PubMed1998

Comparison of electrophysiologic and autonomic tests in sensory diabetic neuropathy.

We examined autonomic function in 46 patients with symmetric sensory non-insulin dependent diabetic neuropathy without autonomic symptoms and 31 age-matched control patients using the composite autonomic scoring scale (CASS) and electrophysiologic examination. The patients were divided into three groups by subjective severity of pain or numbness; 17 had slight pain or numbness, 15 had mild pain or numbness, and 14 had moderate pain or numbness. The patients in the moderate group had the following: a mild reduction in systolic and mean blood pressure (BP) within 1 minute of head-up tilt and a partial recovery after 5 minutes; an excessive fall in early phase II (IIe), an absence of late phase II (IIl) and reduced phase IV beat-to-beat BP responses to Valsalva maneuver (VM); a poor heart rate response to deep breathing; a reduced quantitative sudomotor axon reflex test (QSART) response in distal leg and foot; the highest CASS among the 3 groups; and reduced conduction velocity and amplitude in post-tibial nerve and sural nerve. The mild group had a mild reduction in BP during phase IIe and an absent phase IIl but normal phase IV overshoot during VM; a reduced QSART in the foot; a CASS between the moderate and slight groups; and reduced conduction velocity and amplitude in post-tibial nerve and reduced amplitude in sural nerve. The slight pain group had no abnormalities except for mild cardiovagal dysfunction. CASS gathered from all cases had a significant correlation with amplitude of sural nerve. These results suggest that the patients with symmetric sensory diabetic neuropathy may also have autonomic dysfunction, although they did not have any obvious autonomic symptoms, and that abnormalities in autonomic function parallel changes in somatic function in peripheral nerve. The CASS may be a sensitive tool, similar to the neurophysiologic test, for assessing diabetic neuropathy.

Autonomic Nervous System↗
PubMed1998

In vitro generation of lymphohematopoietic cells from endothelial cells purified from murine embryos.

We have investigated the lymphohematopoietic potentials of endothelial cells (EC) and hematopoietic cells (HPC) sorted from embryos. Expression of VE-cadherin, CD45, and Ter119 was used to distinguish EC (VE-cadherin+CD45-Ter119-) from HPC (VE-cadherin-CD45+). Thus defined, EC population takes up acetylated LDL and coexpresses CD31, Flk1, and CD34. In E9.5 embryos, EC from yolk sac (YS) and the embryo proper generate blood cells, including lymphocytes. Thus, lymphohematopoietic EC do exist in the embryo, and they are generated both in YS and the embryo proper. On the other hand, HPC with lymphopoietic potency appear first in the embryo proper. These findings implicate involvement of multiple environmental cues for acquiring lymphopoietic competency during differentiation of HPC.

Animals↗
PubMed1998

Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa.

Ribozymes, catalytic RNA molecules that cleave a complementary mRNA sequence, have potential as therapeutics for dominantly inherited disease. Twelve percent of American patients with the blinding disease autosomal dominant retinitis pigmentosa (ADRP) carry a substitution of histidine for proline at codon 23 (P23H) in their rhodopsin gene, resulting in photoreceptor cell death from the synthesis of the abnormal gene product. Ribozymes can discriminate and catalyze the in vitro destruction of P23H mutant mRNAs from a transgenic rat model of ADRP. Here, we demonstrate that in vivo expression of either a hammerhead or hairpin ribozyme in this rat model considerably slows the rate of photoreceptor degeneration for at least three months. Catalytically inactive control ribozymes had less effect on the retinal degeneration. Intracellular production of ribozymes in photoreceptors was achieved by transduction with a recombinant adeno-associated virus (rAAV) incorporating a rod opsin promoter. Ribozyme-directed cleavage of mutant mRNAs, therefore, may be an effective therapy for ADRP and also may be applicable to other inherited diseases.

Animals↗