Biomedical subjects

S Nishikawa

Publications and source records attributed to S Nishikawa.

At least 325 records · Page 18Linked to original sources

PubMed1992

Photocoagulation with continuous wave ND:YAG laser for neovascular maculopathy.

Twenty-two eyes in 22 cases of neovascular maculopathy were treated with continuous wave (CW) Nd:YAG laser and followed up for 6 to 24 months. The effect of the treatment was evaluated by repeated visual acuity testing, fundus photography, fluorescein angiography and automated static perimetry. In 91% of the patients visual acuity remained unchanged or improved. Fluorescein angiography revealed that the neovascularization disappeared in 91% of the patients. In 40% of the patients the central visual field showed improvement by automated static perimetry. These results indicate that CW Nd:YAG laser photocoagulation is useful for the treatment of neovascular maculopathy.

Aged↗

PubMed1991

Effect of Steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture.

Despite the importance of germ cells to the survival of species, surprisingly little is known about their embryological origin, proliferation, migration and entry into mitotic arrest or meiosis. Mutations in the murine Dominant White Spotting (W) and Steel genes, which respectively encode the c-kit tyrosine kinase receptor and the c-kit ligand (or Steel factor), impair the development of primordial germ cells (PGCs) in vivo, as well as haematopoietic stem cells and neural crest-derived melanoblasts. Here we use a monoclonal antibody against c-kit tyrosine kinase receptor and recombinant Steel factor to study the c-kit receptor-ligand system in cultured PGCs. In addition, we show that leukaemia inhibitory factor (also known as differentiation inhibitory activity), a factor secreted by STO fibroblasts, can stimulate proliferation of primordial germ cells in vitro.

Animals↗

PubMed1991

Construction of a novel RNA-transcript-trimming plasmid which can be used both in vitro in place of run-off and (G)-free transcriptions and in vivo as multi-sequences transcription vectors.

We have constructed a new transcription system that allows trimming of both 5' and 3'-termini of any RNA transcripts by means of cis-acting ribozyme activities. The vector consists of a promoter, '5' Processing Ribozyme', any DNA template to be transcribed, and '3' Processing Ribozyme' sequences. When the vector possessing T7 promoter was tested in vitro, the transcription efficiency from the circular template was over ten-fold higher than using linearized template (run-off transcription). Further, since uniform RNAs with defined 5'- and 3'-ends can be produced, this strategy complements the conventional run-off transcription. Also the 5'-/3'-trimmed uniform RNA can function as a reporter in elucidating transcription factors and promoter regions in vitro, this strategy can replace the widely used (G)-free transcription (Sawadogo and Roeder (1985) Proc. Natl. Acad. Sci. USA 82, 4394-4398). With this strategy, in addition to the advantage that the template DNA need not be linearized prior to transcription, a cytidine-minus sequence is no longer necessary for quantitative analysis of transcription factors. Since any sequences including those of RNA virus can be inserted between the '5' Processing Ribozyme' and the '3' Processing Ribozyme' sequences, and the entire unit can be inserted into any genes under active transcription, this construct is useful like that of Dzianott and Bujarski ((1989) Proc. Natl. Acad. Sci. USA 86, 4823-4827) for RNA virologists because these strategies provide RNA transcripts without heterologous sequences which may greatly diminish infectivity. Moreover, since the construct can also be used in vivo, multi-transcripts such as trans-acting ribozymes targeted to various sites would be produced by concatenating the entire units in tandem.

Base Sequence↗

PubMed1991

Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule.

The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor for stem cell factor (SCF). The c-kit/SCF signal is expected to have an important role in hematopoiesis. A monoclonal antibody (ACK-2) against the murine c-kit molecule was prepared. Flow cytometric analysis showed that the bone marrow cells that expressed the c-kit molecule (approximately 5%) were B220(B)-, TER119(erythroid)-, Thy1negative-low, and WGA+. A small number of Mac-1(macrophage)+ or Gr-1(granulocyte)+ cells were c-kit-low positive. Colony-forming unit in culture (CFU-C) and day-8 and day-12 CFU-spleen (CFU-S) existed exclusively in the c-kit-positive fraction. About 20% of the Lin(lineage)-c-kit+ cells were rhodamine-123low and this fraction contained more day-12 CFU-S than day-8 CFU-S. On the basis of these findings, murine hematopoietic stem cells were enriched with normal bone marrow cells. One of two and one of four Thy-1lowLin-WGA+c-kit+ cells were CFU-C and CFU-S, respectively. Long-term repopulating ability was investigated using B6/Ly5 congenic mice. Eight and 25 weeks after transplantation of Lin-c-kit+ cells, donor-derived cells were found in the bone marrow, spleen, thymus, and peripheral blood. In peripheral blood, T cells, B cells, and granulocyte-macrophages were derived from donor cells. Injection of ACK-2 into the irradiated mice after bone marrow transplantation decreased the numbers of day-8 and day-12 CFU-S in a dose-dependent manner. Day-8 spleen colony formation was completely suppressed by the injection of 100 micrograms ACK-2, but a small number of day-12 colonies were spared. Our data show that the c-kit molecule is expressed in primitive stem cells and plays an essential role in the early stages of hematopoiesis.

Animals↗

PubMed1991

Non-cognizable ribonucleotide, 2'AMP, binds to a mutant ribonuclease T1 (Y45W) at a new base-binding site but not at the guanine-recognition site.

Complex of a mutant ribonuclease T1 (Y45W) with a non-cognizable ribonucleotide, 2'AMP, has been determined and refined by X-ray diffraction at 1.7 A resolution. The 2'AMP molecule locates at a new base-binding site which is remote from the guanine-recognition site, where 2'GMP was found to be bound. The nucleotide adopts the anti conformation of the glycosidic bond and C3'-exo sugar pucker. There exists a single hydrogen bond between the adenine base and the enzyme, and, therefore, the site found is apparently a non-specific binding site. The results indicate that the binding of 2'AMP to the guanine-recognition site is weaker than that to the new binding site.

Adenosine Monophosphate↗

PubMed1991

The GTP-binding Sar1 protein is localized to the early compartment of the yeast secretory pathway.

SAR1, the yeast gene which encodes a novel type of small GTP-binding protein, has been shown to be required for protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. To further the understanding of the function of its product, a lacZ-SAR1 hybrid gene was constructed and a polyclonal antibody was raised against the hybrid protein. This antibody specifically recognizes the SAR1 gene product (Sar1p) as a 23-kDa protein in the yeast cell lysate. We examined the subcellular localization of Sar1p using this antibody. In wild-type cells, Sar1p was predominantly recovered in a rapidly sedimenting membrane fraction that includes the ER. The soluble form of Sar1p was also detected when the protein was overproduced. Immunofluorescence microscopy with the anti-Sar1p antibody showed perinuclear staining that was exaggerated in the ER-accumulating sec18 mutant. Membrane association of Sar1p was shown to be very light. Sar1p was not extracted from the membrane by treatment with alkaline sodium carbonate, and only 1% deoxycholic acid solubilized Sar1p completely. From these results, we suggest that Sar1p is firmly located on the ER membrane where it regulates the ER-Golgi traffic.

Antibodies↗

PubMed1991

Expression and function of c-kit in hemopoietic progenitor cells.

The expression and function of a receptor tyrosine kinase, c-kit, in the adult bone marrow of the mouse were investigated by using monoclonal antibodies (mAbs) against the extracellular domain of murine c-kit. In adult C57BL/6 mouse, 7.8% of total bone marrow cells express c-kit on their surface. Half of the c-kit+ cells do not express lineage markers including Mac-1, Gr-1, TER-119, and B220, while the remainder coexpress myeloid lineage markers such as Mac-1 and Gr-1. After c-kit+ cells were removed from the bone marrow cell preparation, hemopoietic progenitor cells reactive to IL-3, GM-CSF, or M-CSF and also those which give rise to spleen colonies in irradiated recipients disappeared almost completely. Thus, most hemopoietic progenitors in the adult bone marrow express c-kit. To investigate whether or not c-kit has any role in the hemopoiesis of adult bone marrow, we took the advantage of one of the anti-c-kit mAbs that can antagonize the function of c-kit. As early as two days after the injection of 1 milligram of an antagonistic antibody, ACK2, almost all hemopoietic progenitor cells disappeared from the bone marrow, which eventually resulted in the absence of mature myeloid and erythroid cells in the bone marrow. These results provide direct evidence that c-kit is an essential molecule for constitutive intramarrow hemopoiesis, especially for the self-renewal of hemopoietic progenitor cells at various stages of differentiation.

Animals↗

PubMed1991

Support of early B-cell differentiation in mouse fetal liver by stromal cells and interleukin-7.

We compared the development of B-cell progenitors with that of myeloid progenitors in fetal liver cells at various gestational ages. Day 12 to 14 fetal liver cells did not form pre-B-cell colonies. Pre-B-cell colonies were developed from day 15 fetal liver cells. The incidence of colonies increased with increases in gestational age and reached a maximum on days 18 to 19. In contrast, the incidence of myeloid colonies formed in the presence of interleukin-3 (IL-3) and erythropoietin did not change significantly during days 13 to 21 of gestation. After coculturing day 13 fetal liver cells with IL-7-producing stromal cell line ST-2, they could respond to IL-7 and proliferate. Analysis of the phenotypes showed that day 13 fetal liver cells were B220-, IgM-, while culturing day 13 fetal liver cells with ST-2 and untreated day 18 fetal liver cells contained the population of B220+ cells. Even in the presence of IL-7-defective stromal cell line FLS-3, IL-7-responsive cells could be induced from day 13 fetal liver cells. IL-7 acted on B220+ cells and induced pre-B-cell colonies that contained IgM+ cells in the methylcellulose culture. IL-7 mRNA was expressed in days 13 and 18 fetal liver cells but not in pre-B cells or adult liver cells. From these findings, it is suggested that stromal cells or stromal-derived factors but not IL-7 were required for the differentiation from B220- cells to B220+ cells. In the second stage, B220+, IgM- cells proliferated and some of them differentiated to IgM+ cells in the presence of IL-7 alone. The two-step model can apply to in vivo early B lymphopoiesis.

Animals↗

PubMed1991

Rate limiting P-O(5') bond cleavage of RNA fragment: ab initio molecular orbital calculations on the base-catalyzed hydrolysis of phosphate.

In order to examine the energetics in base-catalyzed hydrolysis of RNA, a tentative pentacoordinated intermediate (3) has been characterized by molecular orbital calculations. Ab initio studies at the level of 3-21G* indicate that, under the Cs symmetry restricted conditions, the P-O(2) bond possessing antiperiplanar (app) lone pair electrons (Ip) on the equatorial oxygen (O(3)) can be cleaved with almost no barrier (TS1 transition state; 0.08 kcal mol-1), from the pentacoordinated intermediate (3) of base-catalyzed hydrolysis of phosphate, compared to the P-O(5) bond (TS2 transition state; 28.9 kcal mol-1) which lacks app lp assistance from O(3). The dianionic intermediate, however, loses the TS1 transition state thus its property as an intermediate when the Cs restriction is removed. The analysis of the entire potential energy surface enables us to conclude that, in a related system examined by Lim and Karplus [1990) J. Am. Chem. Soc., 112, 5872-5873) for attack by OH- on ethylene phosphate monoanion, the TS1 transition state had also been lost and thus no intermediate had been found. These results further support our earlier conclusions (Taira et al. (1990) Protein Engineering, 3, 691-701) of rate limiting transition state possessing extended P-O(5') bond breaking character (the TS2 transition state) in the base-catalyzed hydrolysis of RNA. Finally, although the lack of 2',3' -migration of phosphate moieties in basic condition appears to be in accord with the short-lived intermediate, it really does not prove the absence of the intermediate. The detail will be discussed in the text.

Hydrolysis↗

PubMed1991

Combination of Trp and Glu residues for recognition of mRNA cap structure. Analysis of m7G base recognition site of human cap binding protein (IF-4E) by site-directed mutagenesis.

Four mutants of the human cap binding protein (hCBP), in which Trp-102, Glu-103, Asp-104 or Glu-105 was changed to the aliphatic Leu or Ala, were prepared, and their cap binding abilities were examined. Cap binding abilities of two mutants, W102L (Trp-102----Leu) and E105A (Glu-105----Ala), were significantly decreased in comparison with the wild-type hCBP. This result suggests that Trp-102 and Glu-105 are both necessary for the cap binding, and the most probable binding mode with the m7G of cap structure is the combination of the stacking by Trp-102 and the hydrogen-bond pairing by Glu-105, as was already proposed from the model studies.

Amino Acid Sequence↗

PubMed1991

Exon skipping by mutation of an authentic splice site of c-kit gene in W/W mouse.

The murine mutation dominant white spotting (W) is in the proto-oncogene, c-kit. The receptor tyrosine kinase encoded by this gene has pleiotropic effects on murine development including hemopoietic cells, pigment cells, and germ cells. In this study, mutation in W homozygous mouse was identified as a single base substitution (GT----AT) at the 5'-splice donor site of the exon which encodes the transmembrane domain. Two types of aberrant exon skipping resulted from this mutation, occurred in a tissue specific manner. Either transcript lost the exon coding for transmembrane region and therefore the product might not be functional for signal transduction. Any unusual cryptic splice sites were not activated by this mutation as beta-globin gene in beta-thalassaemia. In addition, twelve base pair sequence of the 3'-end of the exon prior to the exon coding for transmembrane domain was found to be alternatively spliced. These findings should provide the genetic base for not only the receptor function but the splicing mechanism.

Animals↗

PubMed1991

Specific depletion of the B-cell population induced by aberrant expression of human interferon regulatory factor 1 gene in transgenic mice.

Interferons (IFNs) are well known both as antiviral proteins and as potent regulators of cell growth and differentiation. In fact, IFNs inhibit growth of various normal and transformed cell types. Previously, a nuclear factor, IRF-1 (interferon regulatory factor 1), which binds to type I IFN and some IFN-inducible gene promoters, was identified and cloned. Since the IRF-1 gene is both virus and IFN inducible, an intriguing issue is raised as to whether the IRF-1 gene is functioning in IFN-mediated regulation of cell growth and differentiation. In this study, we generated transgenic mice carrying the human IRF-1 gene linked to the human immunoglobulin heavy-chain enhancer. In the transgenic mice, all the lymphoid tissues examined showed a dramatic reduction in the number of B lymphocytes (B cells). Preparation and analysis of bone marrow cells from the chimeric mice indicated that the bone marrow is the effective site for specific depletion of the B-cell population. In fact, transgenic bone marrow cells cocultured with a bone marrow-derived stromal cell line revealed an altered B-cell maturation pattern.

Animals↗

PubMed1991

Response of newly established mouse myeloid leukemic cell lines to MC3T3-G2/PA6 preadipocytes and hematopoietic factors.

Some mouse myeloid leukemias induced by X-irradiation and serially transplanted into syngenic mice do not proliferate in vitro even in the presence of hematopoietic factors. To examine whether such leukemic cells can proliferate in response to stromal cells, we cocultured them with MC3T3-G2/PA6 (PA6) preadipocytes, cells that can support the growth of hematopoietic stem cells. All leukemias developed into in vitro cell lines, showing a dependence on contact with the PA6 cells. Two cell lines responded to none of the known hematopoietic factors including interleukin-3 (IL-3), IL-4, IL-5, IL-6, GM-CSF, G-CSF, M-CSF, and Epo. These results demonstrate that the mechanism of the action of PA6 cells is different from that of any of the known hematopoietic factors, and that, because these two leukemic cell lines retained the ability to grow in vivo, responsiveness to the known hematopoietic factors is not essential for the leukemic cell growth in vivo. Furthermore, all leukemic cell lines could respond also to the preadipocytes fixed with formalin, paraformaldehyde, or glutaraldehyde, suggesting that some molecule(s) associated with the surface of PA6 cells or with extracellular matrix secreted by the preadipocytes is responsible for the leukemic cell growth.

Adipose Tissue↗

PubMed1991

The c-kit-encoded tyrosine kinase regulates the proliferation of early pre-B cells.

A monoclonal antibody (mAb; ACK2) recognizing the extracellular domains of the c-kit-encoded tyrosine kinase has been employed to demonstrate that c-kit is involved in B lymphocyte development. The c-kit-encoded tyrosine kinase is expressed on the surface of normal DHJH-rearranged murine pre-B cell clones which proliferate continuously at that stage in vitro on stromal cells and in the presence of recombinant interleukin 7. These pre-B cell clones, capable of differentiation to surface immunoglobulin-positive B cells in vitro and in vivo, are inhibited by the mAb in their proliferation while remaining capable of differentiation to surface immunoglobulin-positive B cells. Stimulation of mature B cells by mitogens is unimpaired by the mAb. This indicates that c-kit regulates early antigen-independent, but not late antigen-dependent, B cell development.

Animals↗

PubMed1991

In utero manipulation of coat color formation by a monoclonal anti-c-kit antibody: two distinct waves of c-kit-dependency during melanocyte development.

Previous studies on mice bearing various mutations within the c-kit gene, dominant white spotting (W), indicate the functional role of this tyrosine kinase receptor in the development of melanocytes, germ cells and hematopoietic cells. Despite the availability of mice defective in the c-kit gene and a respectable understanding of the molecular nature of c-kit, however, it is not clear at what stage of gestation c-kit is functionally required for the development of each of these cell lineages. To address this question, we have used a monoclonal anti-c-kit antibody, ACK2, as an antagonistic blocker of c-kit function to interfere with the development of melanocytes during embryonic and postnatal life. ACK2 injected intradermally into pregnant mice entered the embryos where it blocked the proper development of melanocytes. This inhibitory effect was manifested as coat color alteration in the offspring. Furthermore, ACK2 injection also altered the coat color of neonatal and adult mice. Based on the coat color patterns produced by ACK2 administration at various stages before or after birth, the following conclusions are drawn: (i) during mid-gestation, c-kit is functionally required during a restricted period around day 14.5 post-coitum when a sequence of events leading to melanocyte entry into the epidermal layer occurs; (ii) during postnatal life, c-kit is required for melanocyte activation which occurs concomitantly with the hair cycle which continues throughout life after neonatal development of the first hair.

Animals↗

PubMed1991

Differentiation of growth signal requirement of B lymphocyte precursor is directed by expression of immunoglobulin.

During B cell differentiation, at least three stages can be defined in terms of their growth signal requirement by using two different growth signals, which are recombinant interleukin 7 (IL-7) and a stromal cell clone PA6 which does not produce IL-7; first a PA6 dependent stage, second a PA6 + IL-7 dependent stage and third an IL-7 dependent stage. In order to test the possibility that this differentiation of growth signal requirement is controlled by the expression of functional immunoglobulin molecules, we have investigated the frequencies of PA6 + IL-7 dependent and IL-7 dependent cells which are present in the bone marrow of either mu-chain or kappa-chain gene transgenic mice. In a mu-chain gene transgenic mouse, the frequency of PA6 + IL-7 dependent cells is selectively reduced, while that of IL-7 dependent cells is selectively reduced in a kappa-chain gene transgenic mouse. This result suggests that expression of a functional mu-chain gene drives PA6 + IL-7 dependent cells to differentiate into the subsequent IL-7 dependent stage. Likewise, when mu-chain positive IL-7 dependent cells express a functional light-chain gene, their growth signal requirement changes into an IL-7 unreactive stage.

Animals↗

PubMed1991

Molecular and cellular aspects of early B-cell development.

The molecular mechanisms underlying pre-B cell growth and immunoglobulin gene rearrangement have been actively investigated. Some growth factors for B-cell precursors as well as some genes activating V(D)J recombination have been identified. Furthermore, the molecular structure and signaling capacity of the surface immunoglobulin of pre-B cells have been characterized.

Animals↗

PubMed1991

Effects of parathyroid hormone, 1,25-dihydroxyvitamin D3, and prostaglandin E2 on alkaline phosphatase activity in cultured dental pulp and gingiva cells of bovine calf.

The effects of parathyroid hormone (PTH), 1,25-dihydroxyvitamin D3, and prostaglandin E2 (PGE2) on alkaline phosphatase activity on cultured dental pulp and gingiva cells of bovine calf were compared. In pulp cells, PTH, 1,25-dihydroxyvitamin D3, and PGE2 significantly increased alkaline phosphatase activity, but no increase in the enzyme activity by these factors was observed in gingiva cells. Dibutyryl cAMP also increased alkaline phosphatase activity in both types of cell, but the increase in pulp cells was greater than that in gingiva cells. Treatment of the cultured pulp cells with PTH or PGE2 significantly increased the intracellular cAMP content. These results suggest that calciotropic factors such as PTH, 1,25-dihydroxyvitamin D3, and PGE2 may be involved in the differentiation of dental pulp cells and that some of these effects (those of PTH and PGE2) are mediated by cAMP.

Alkaline Phosphatase↗