Biomedical subjects

S Nishikawa

Publications and source records attributed to S Nishikawa.

At least 19 recordsLinked to original sources

PubMed1999

Detailed analysis of stem I and its 5' and 3' neighbor regions in the trans-acting HDV ribozyme.

To determine the stem I structure of the human hepatitis delta virus (HDV) ribozyme, which is related to the substrate sequence in the trans -acting system, we kinetically studied stem I length and sequences. Stem I extension from 7 to 8 or 9 bp caused a loss of activity and a low amount of active complex with 9 bp in the trans -acting system. In a previous report, we presented cleavage in a 6 bp stem I. The observed reaction rates indicate that the original 7 bp stem I is in the most favorable location for catalytic reaction among the possible 6-8 bp stems. To test base specificity, we replaced the original GC-rich sequence in stem I with AU-rich sequences containing six AU or UA base pairs with the natural +1G.U wobble base pair at the cleavage site. The cis -acting AU-rich molecules demonstrated similar catalytic activity to that of the wild-type. In trans -acting molecules, due to stem I instability, reaction efficiency strongly depended on the concentration of the ribozyme-substrate complex and reaction temperature. Multiple turnover was observed at 37 degreesC, strongly suggesting that stem I has no base specificity and more efficient activity can be expected under multiple turnover conditions by substituting several UA or AU base pairs into stem I. We also studied the substrate damaging sequences linked to both ends of stem I for its development in therapeutic applications and confirmed the functions of the unique structure.

Catalysis↗

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↗

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↗

PubMed1998

Mechanism of endothelin-1 release from endothelial cells in pregnancy-induced hypertension.

We investigated the mechanism of vasoconstrictor-induced endothelin-1 (ET-1) release from human umbilical vein endothelial cells (HUVECs) in serum from women with pregnancy-induced hypertension (PIH). We obtained serum samples from seven women with PIH, seven healthy nonpregnant women (NP), and seven normal pregnant women (NPIH). ET-1 and inositol 1,4,5-trisphosphate (IP3) were assayed by ET-1 ELISA and an IP3 3H assay system, respectively. ET-1 release from HUVECs incubated with 10% serum (NP, NPIH, and PIH) was greater than that without human serum. Angiotensin II (Ang II)- and epinephrine (Epi)-induced ET-1 release were significantly increased by PIH serum. IP3 production in HUVECs incubated with 10% serum (NP, NPIH, and PIH) was greater than that without human serum. Ang II- or Epi-induced IP3 production in HUVECs incubated with PIH serum was increased but not significantly compared to that with other sera. Our results suggest that increased ET-1 release from HUVECs incubated with human serum may be mediated by IP3 production, but that Ang II- or Epi-induced ET-1 release from HUVECs incubated with PIH serum may be mediated by another mechanism.

Endothelin-1↗

PubMed1998

Systemic dissemination by intrarectal infection with Listeria monocytogenes in mice.

Orally ingested Listeria monocytogenes is known to penetrate into Peyer's patches (PP) and translocate to the spleen and liver. Herein, extraintestinal dissemination of the bacterium independent of PP was investigated. Dissemination of Listeriae to the spleen and liver was observed in intrarectally infected mice as well as in intragastrically infected animals in spite that no Listeriae were detected in the small intestines of mice infected intrarectally. Decreased numbers of intestinal intraepithelial lymphocytes (iIEL) and increased numbers of lymphocytes in the contents of the small and large intestines were observed after intragastric infection and in the large intestine after intrarectal infection, giving the assumption that the leakage of iIEL caused by injury of epithelial layers in intestines might occur during infection. These results suggest that L. monocytogenes might be able to disseminate through small and large intestines in part by a PP-independent mechanism.

Animals↗

PubMed1998

Neuropeptides in the livers of mice during bacterial infections.

Neuropeptides such as substance P (SP) and vasoactive intestinal peptide (VIP) are known to act as immunomodulators. We investigated the induction of SP and VIP in the livers of mice infected with Listeria monocytogenes or injected with Tsukamurella paurometabolum. VIP was detected in the livers of mice after L. monocytogenes infection by an immunohistochemical technique and preproVIP mRNA, which was detected by reverse transcription-polymerase chain reaction (PCR), was induced post infection. However, no SP was detected. In contrast, SP, but not VIP was detected within granulomas in the livers of T. paurometabolum-injected mice, suggesting VIP and SP might be selectively induced in the liver by different bacterial infections.

Actinomycetales↗

PubMed1998

Cloning and sequencing of the Sphingomonas (Pseudomonas) paucimobilis gene essential for the O demethylation of vanillate and syringate.

Sphingomonas (Pseudomonas) paucimobilis SYK-6 is able to grow on 5,5'-dehydrodivanillic acid (DDVA), syringate, vanillate, and other dimeric model compounds of lignin as a sole carbon source. Nitrosoguanidine mutagenesis of S. paucimobilis SYK-6 was performed, and two mutants with altered DDVA degradation pathways were isolated. The mutant strain NT-1 could not degrade DDVA, but could degrade syringate, vanillate, and 2,2',3'-trihydroxy-3-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA). Strain DC-49 could slowly assimilate DDVA, but could degrade neither vanillate nor syringate, although it could degrade protocatechuate and 3-O-methylgallate. A complementing DNA fragment of strain DC-49 was isolated from the cosmid library of strain SYK-6. The minimum DNA fragment complementing DC-49 was determined to be the 1.8-kbp insert of pKEX2.0. Sequencing analysis showed an open reading frame of 1,671 bp in this fragment, and a similarity search indicated that the deduced amino acid sequence of this open reading frame had significant similarity (60%) to the formyltetrahydrofolate synthetase of Clostridium thermoaceticum.

Amino Acid Sequence↗