Search PubMed⌕ Search

Biomedical subjects

S Nishikawa

Publications and source records attributed to S Nishikawa.

At least 181 records · Page 10Linked to original sources
PubMed1997

In vitro analysis of potency restriction during epiblast differentiation.

During mammalian gastrulation, posterior part of primitive ectoderm differentiates into subsets of mesoderm cells, the most proximal portion of which subsequently gives rise to vascular endothelial cells and hematopoietic cells. To analyze this process in which the potency of multipotent epiblast cells is restricted progressively into hematopoietic cell lineages, we have established a culture system that allows differentiation of epiblast cells as well as embryonic stem cells into hematopoietic cells. Using this culture system, we showed that all parts of epiblast tissues at early streak and late bud stages preserved hematogenic potency, while it was lost from anterior region at early head fold stages. However, this loss of hematogenic potency in the anterior region of the head fold stage embryo was reinduced by addition of activin in the culture. Moreover, in order to detect transitory stages of mesoderm cells that are the direct progeny of multipotent epiblast cells, we developed three monoclonal antibodies that are able to define distinct subsets of mesoderm cells in the gastrulating embryo. These results are discussed from a view of cell-mass based commitment.

Animals↗
PubMed1997

Specific RNA aptamers to NS3 protease domain of hepatitis C virus.

In order to isolate RNA aptamers that bind specifically to NS3 protease domain (delta NS3) of hepatitis C virus, we carried out in vitro selection procedure using RNA pool that had 30 N random core region. After repeating nine cycles of selections and amplifications, a pool of RNAs that bind specifically to the delta NS3 were selected. A comparative analysis of 45 clones that were isolated from 9th cycle revealed three main classes that contain the conserved loop sequences GANUGGGAC. Moreover, the predominant class of aptamer (class I and III) appear to inhibit the protease activity efficiently.

Base Sequence↗
PubMed1996

The protective role of endogenous cytokines in host resistance against an intragastric infection with Listeria monocytogenes in mice.

It has been demonstrated that endogenous cytokines including gamma-interferon (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) play a protective role but that IL-4 plays a detrimental role in systemic Listeria monocytogenes infection in mice. The diverse roles of IL-10 have been reported in antilisterial resistance. In this paper, we studied the role of endogenous cytokines in host resistance against an intragastric infection with L. monocytogenes in mice. The expression of cytokine mRNAs including IFN-gamma, TNF-alpha, IL-4, IL-6, or IL-10, which were amplified by reverse transcription-PCR, was observed in intestinal intraepithelial lymphocytes irrespective of L. monocytogenes infection. Increased numbers of L. monocytogenes were detected in the ileal contents of infected mice which received monoclonal antibodies (mAbs) against IFN-gamma, TNF-alpha, IL-4, IL-6, or IL-10. By contrast, mAbs against IL-4 or IL-6 showed little effect on the growth of L. monocytogenes in the mesenteric lymph nodes (MLNs), spleen, and liver, but anti-IFN-gamma mAb and anti-TNF-alpha mAb suppressed the defense in these organs. Anti-IL-10 mAb enhanced bacterial elimination from the MLNs but not from the spleen or liver. These results suggest that the role of endogenous cytokines may differ between systemic and intestinal defenses.

Animals↗
PubMed1996

Preferential proliferation of murine colony-forming units in culture in a chemically defined condition with a macrophage colony-stimulating factor-negative stromal cell clone.

The establishment of culture conditions that selectively support hematopoietic stem cells is an important goal of hematology. In this study, we investigated the possibility of using for this purpose a defined medium, mSFO2, which was developed for stromal cell-dependent bone marrow cultures. We found that a combination of epidermal growth factor (EGF), the OP9 stromal cell line, which lacks macrophage colony-stimulating factor, recombinant stem cell factor, and the chemically defined medium mSFO2 provides a microenvironment where c-Kit+ Thy-1+/lo Mac-1+/lo B220- TER119- common beta + IL-2R gamma + gp130+ cells are selectively propagated from normal, unfractionated bone marrow cells. This cell population produced an in vitro colony at a very high efficiency (50%), whereas it has only limited proliferative ability in the irradiated recipient. Thus, the cells selected in this culture condition might represent colony-forming units in culture (CFU-c) with short-term reconstituting ability. Transferring this cell population into medium containing differentiation signals resulted in the rapid production of mature myelomonocytic and B cell lineages in vitro and in vivo. The fact that a similar culture condition was created by erb-B2-transduced OP9 in the absence of EGF indicated that EGF exerts its effect by acting on OP9 rather than directly on CFU-c. These results suggested that the balance between self-renewal and differentiation of CFU-c can be regulated by extra-cellular signals.

Animals↗
PubMed1996

Enteric neurons express Steel factor-lacZ transgene in the murine gastrointestinal tract.

The cells that express Steel factor (SLF) in the gastrointestinal (GI) tract were studied using SLF-lacZ transgenic mice. Expression, detected by beta-galactosidase histochemistry, was evident in cells between the circular and longitudinal muscle layers in the GI tract. Double staining with antibodies specific for the neural markers, PGP 9.5, MAP2 and c-Ret, showed that SLF-lacZ positive cells were enteric neurons. Enteroglia did not express SLF-lacZ. The distribution of expressing cells was complimentary to the expression of c-Kit in myenteric interstitial cells.

Animals↗
PubMed1996

Constructing an efficient trans-acting genomic HDV ribozyme.

We have engineered a genomic HDV ribozyme to construct several trans-acting ribozymes for use in trans to cleave target RNAs. Among the 10 different combinations attempted, only HDV88-Trans had cleavage activity on the 13-nucleotide substrate, R13, in vitro. To improve the cleavage efficiency, at least in vitro, of the HDV88-Trans ribozyme (kclv = 0.022 min(-1)), we have constructed several variants that differ in forming stem II (length) in the pseudoknot secondary structure model. When cleavage rate constants were analyzed and compared among variants of HDV88-Trans, HDV88-Trans-4 yielded kclv = 1.7 min(-1). HDV88-Trans-4 thus represents the highest active genomic HDV ribozyme that functions in trans thus far constructed, and has activity under physiological conditions (pH 7.1 at 37 degrees C with 1 mM of MgCl2).

Base Sequence↗
PubMed1996

Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1.

The chemokines are a large family of small, structurally related cytokines. The physiological importance of most members of this family has yet to be elucidated, although some are inducible inflammatory mediators that determine leukocyte chemotaxis. Pre-B-cell growth-stimulating factor/stromal cell-derived factor-1 (PBSF/SDF-1) is a member of the CXC group of chemokines PBSF/SDF-1 stimulates proliferation of B-cell progenitors in vitro and is constitutively expressed in bone-marrow-derived stromal cells. Here we investigate the physiological roles of PBSF/SDF-1 by generating mutant mice with a targeted disruption of the gene encoding PBSF/SDF-1. We found that mice lacking PBSF/SDF-1 died perinatally and that although the numbers of B-cell progenitors in mutant embryos were severely reduced in fetal liver and bone marrow, myeloid progenitors were reduced only in the bone marrow but not in the fetal liver, indicating that PBSF/SDF-1 is responsible for B-cell lymphopoiesis and bone-marrow myelopoiesis. In addition, the mutants had a cardiac ventricular septal defect. Hence, we have shown that the chemokine PBSF/SDF-1 has several essential functions in development.

Animals↗
PubMed1996

Dependence on Mg2+ ions of the activities of dimeric hammerhead minizymes.

A minizyme is a hammerhead ribozyme with short oligonucleotide linkers instead of stem/loop II. In a previous study we demonstrated that a minizyme with high-level activity forms a dimeric structure with a common stem II [Amontov and Taira (1996) J. Am. Chem. Soc. 118, 1624-1628]. We now demonstrate that the stability of the dimeric structure is influenced by Mg2+ ions. We found that the dependence on Mg2+ ions of the activity of homodimeric minizyme (a dimer with two identical binding sites) has composite biphasic characteristics. When the concentration of Mg2+ ions reached a specific critical level, the dependence on the concentration of Mg2+ ions lost its tendency to reach a plateau. In the case of the heterodimeric minizyme (a dimer with two different binding sites), we investigated the kinetic behavior of two different forms of the dimer, namely, free dimer and the complex of the dimer with an uncleavable substrate. The kinetic behavior of the free heterodimer was very similar to that of the homodimeric minizyme. In contrast, in the presence of the uncleavable substrate at a concentration as high as that of the minizyme, the curve for the dependence on Mg2+ ions showed normal saturation kinetics. While, at low concentrations of Mg2+ ions, the activity of the heterodimers was much higher when the dimeric structure was stabilized by the presence of the uncleavable substrate, at high concentrations of Mg2+ ions, this difference in activity became less and less significant. Thus, high concentrations of Mg2+ ions were able to stabilize the dimeric minizymes in the absence of the uncleavable substrate.

Base Sequence↗
PubMed1996

Selection in vitro of trans-acting genomic human hepatitis delta virus (HDV) ribozymes.

In an effort to identify the functional structure as well as new active variants of the trans-acting genomic ribozyme of human hepatitis delta virus (HDV), we applied an in vitro selection procedure. A total of 14 rounds of selection and amplification was repeated and various mutant ribozymes in G10 and G14 pools analyzed. Active ribozymes which were isolated in the present study (from G10 and G14) all possessed conserved bases (that were identified earlier) in the cis-acting molecule. A dominant clone G10-68 variant was accumulated in generation 14. Interestingly, when base substitutions were analyzed in G10-68 variant, we found that this variant appears to be close to antigenome-like HDV ribozyme molecule. Further investigations of G10-68 confirmed that each mutated base was the most appropriate nucleotide at every position of the HDV ribozyme.

Base Sequence↗
PubMed1996

Magnesium-mediated conversion of an inactive form of a hammerhead ribozyme to an active complex with its substrate. An investigation by NMR spectroscopy.

The effects of magnesium ions on a 32-mer ribozyme (R32) were examined by high resolution NMR spectroscopy. In solution, R32 (without its substrate) consisted of a GAAA loop, stem II, a non-Watson-Crick 3-base pair duplex and a 4-base pair duplex that included a wobble G:U base pair. When an uncleavable substrate RNA (RdC11) was added to R32 without Mg2+ ions, a complex did not form between R32 and RdC11 because the substrate recognition regions of R32 formed intramolecular base pairs (the recognition arms were closed). By contrast, in the presence of Mg2+ ions, the R32-RdC11 complex was formed. Moreover, titration of mixtures of R32 and RdC11 with Mg2+ ions also induced the ribozyme-substrate interaction. Elevated concentrations (1.0 M) of monovalent Na+ ions could not induce the formation of the R32-RdC11 complex. These data suggest that Mg2+ ions are not only important as the true catalysts in the function of ribozyme-type metalloenzymes, but they also induce the structural change in the R32 hammerhead ribozyme that is necessary for establishment of the active form of the ribozyme-substrate complex.

Base Composition↗
PubMed1996

Interleukin 2 (IL-2) and interleukin 7 (IL-7) reciprocally induce IL-7 and IL-2 receptors on gamma delta T-cell receptor-positive intraepithelial lymphocytes.

In this study, we describe the interaction between cytokine and cytokine receptor (R) for the activation and proliferation of gamma delta T-cell receptor-positive T cells (gamma delta T cells). gamma delta T cells isolated from murine intestinal intraepithelial lymphocytes (IELs) were separated into gamma delta (Dim) and gamma delta (Bright) fractions according to the intensity of gamma delta T-cell receptor expression. The gamma delta T cells express low levels of IL-2R and IL-7R as shown by flow cytometry and reverse transcriptase-PCR analysis, whereas gamma delta (Bright) T cells did not express either receptor. Our study also revealed that recombinant marine (rm)IL-2 and rmIL-7 reciprocally induced high expressions of IL-7R and IL-2R, respectively, on gamma delta (Dim) T cells but not on gamma delta (Bright) cells. Thus, treatment of gamma delta (Dim) T cells with rmIL-2 and rmIL-7 resulted in high proliferative responses, whereas gamma delta (Bright) T cells did not respond to these two cytokines. The sources of these two cytokines for gamma delta T cells were neighboring epithelial cells (IL-7) and alpha beta T cells (IL-2 and IL-7). Cytokine signaling by IL-2 and IL-7 from alpha beta T cells and epithelial cells was necessary for the expression of IL-7R and IL-2R, respectively, on a subset of gamma delta T cells (e.g., gamma delta (Dim) T cells) in mucosa-associated tissue for subsequent activation and cell division.

Animals↗
PubMed1996

High-resolution NMR study of a GdAGA tetranucleotide loop that is an improved substrate for ricin, a cytotoxic plant protein.

Ricin is a cytotoxic plant protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond at position A4324 in eukaryotic 28S rRNA. Recent studies showed that a four-nucleotide loop, GAGA, can function as a minimum substrate for ricin (the first adenosine corresponds to the site of depurination). We previously clarified the solution structure of this loop by NMR spectroscopy [Orita et al. (1993) Nucleic Acids Res. 21, 5670-5678]. To elucidate further details of the structural basis for recognition of its substrate by ricin, we studied the properties of a synthetic dodecanucleotide, r1C2U3C4A5G6dA7G8A9U10G11A12G (6dA12mer), which forms an RNA hairpin structure with a GdAGA loop and in which the site of depurination is changed from adenosine to 2'-deoxyadenosine. The N-glycosidase activity against the GdAGA loop of the A-chain of ricin was 26 times higher than that against the GAGA loop. NMR studies indicated that the overall structure of the GdAGA loop was similar to that of the GAGA loop with the exception of the sugar puckers of 6dA and 7G. Therefore, it appears that the 2'-hydroxyl group of adenosine at the depurination site (6A) does not participate in the recognition by ricin of the substrate. Since the 2'-hydroxyl group can potentially destabilize the developing positive charge of the putative transition state intermediate, an oxycarbonium ion, the electronic effect may explain, at least in part, the faster rate of depurination of the GdAGA loop compared to that of GAGA loop. We also show that the amino group of 7G is essential for substrate recognition the ricin A-chain.

Base Sequence↗
PubMed1996

Neural and skin cell-specific expression pattern conferred by steel factor regulatory sequence in transgenic mice.

We have produced transgenic mice expressing a lacZ reporter gene under the control of a fragment of a Steel factor (SLF). The function of this gene is essential for the development of hematopoietic cells, germ cells, melanocytes and pacemaker cells of the intestine. The expression of the transgene, containing 2 kb DNA 5' regulatory sequence, was restricted to neural and skin tissues in appropriate spatial and temporal pattern compared with endogenous SLF mRNA expression. This indicates that the regulatory elements necessary for the neural and skin specific expression are present in this 2 kb DNA sequence, although strong position-dependence of transgene expression was observed. As we could not detect transgene expression in hematopoietic tissues and germ cells after extending 10 kb upstream, elements important for these organs must reside in other regions. Our results indicate that neural crest derived enteric ganglion cells provide SLF to the neighboring pace maker cells expressing c-kit, the receptor for SLF. Cells expressing the transgene in the intestine are ganglion cells derived from neural crest since homozygosity for the lethal spotting (Is) mutation results in loss of such ganglion cells in transgenic mice. We have also shown that the dermal papillae of the hair follicle expresses the transgene, suggesting its roles to support the c-kit dependent growth and development of melanocytes in the hair follicle.

Animals↗
PubMed1996

Molecular cloning of a novel type I receptor serine/threonine kinase for the TGF beta superfamily from rat brain.

Growth factors belonging to the TGF beta superfamily bind to and signal through a receptor complex comprising two transmembrane serine/threonine kinases, called type I and type II. Each receptor is responsible for the signaling of the individual TGF beta superfamily members. So far, five type II and six type I receptors have been cloned from mammalian sources. We report here the molecular cloning of a novel type I receptor serine/threonine kinase, ALK7 (activin receptor-like kinase 7), from rat brain. ALK7 shows a significant sequence similarity with TGF beta RI and ActRIB in the intracellular kinase domain and is quite distinct from other type I receptors in the extracellular domain. ALK7 mRNA is expressed in embryonic and in adult rat brain, where it was localized in superficial layers of the forebrain, the CA3 pyramidal subfield of hippocampus, the basal ganglia, the thalamus, and the cerebellar cortex. The functionality of the receptor was demonstrated by the identification of a constitutively active point mutant of ALK7 that activates the TGF beta/activin-responsive reporter without any ligand stimulation. Although the endogenous ligand for ALK7 has yet to be identified, its extensive anatomic distribution in brain, gut, spleen, and lung suggests important roles for this orphan receptor.

Activin Receptors↗
PubMed1996

Frontal mental theta wave recorded simultaneously with magnetoencephalography and electroencephalography.

Frontal mental theta waves were recorded simultaneously with magnetoencephalography (MEG) and electroencephalography (EEG) in normal subjects. MEG analyses demonstrated that theta burst activities distribute widely in the frontal cortices of both the cerebral hemispheres during concentration on mental calculation. On the other hand, EEG showed the theta activities seemed maximal on the frontal midline part (Fz), as if they might have been generated mainly in the frontal midline cortical area.

Adult↗
PubMed1996

PDGF mediates a neuron-astrocyte interaction in the developing retina.

Astrocytes invade the developing retina from the optic nerve head, over the axons of retinal ganglion cells (RGCs). RGCs express the platelet-derived growth factor A-chain (PDGF-A) and retinal astrocytes the PDGF alpha-receptor (PDGFR alpha), suggesting that PDGF mediates a paracrine interaction between these cells. To test this, we inhibited PDGF signaling in the eye with a neutralizing anti-PDGFR alpha antibody or a soluble extracellular fragment of PDGFR alpha. These treatments inhibited development of the astrocyte network. We also generated transgenic mice that overexpress PDGF-A in RGCs. This resulted in hyperproliferation of astrocytes, which in turn induced excessive vasculogenesis. Thus, PDGF appears to be a link in the chain of cell-cell interactions responsible for matching numbers of neurons, astrocytes, and blood vessels during retinal development.

Animals↗