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Biomedical subjects

S Nishikawa

Publications and source records attributed to S Nishikawa.

At least 19 recordsLinked to original sources
PubMed1995

Overexpression of retinoic acid receptor alpha suppresses myeloid cell differentiation at the promyelocyte stage.

Retinoic acid receptor (RAR) alpha is required to heterodimerize with retinoid X receptor (RXRs) in order to regulate myeloid differentiation. If so, it is expected that overexpression of normal RAR alpha may perturb the RAR alpha/RXR heterodimer formation and also the differentiation of myeloid cells. We have described here the morphology and the RA response of human RAR alpha cDNA transduced murine bone marrow cells using a retroviral vector. Most of RAR alpha transduced cells displayed promyelocyte like morphology and their proportion of c-kit expressing population was increased remarkably compared with the control (Neor gene transduced cells). Furthermore, this morphology was observed even after these cells were brought into the semisolid culture containing IL-3 alone. Interestingly, immature RAR alpha transduced cells differentiated into mature granulocytes under the condition of the high concentration of RA(10(-6) M). We did not observe any effect of RAR alpha on monocytes. These results indicate that overexpression of normal RAR alpha is sufficient for inducing maturation arrest of myeloid cell lineage that is similar to the phenotype found in the acute promyelocytic leukemia bearing PML-RAR alpha translocation.

3T3 Cells↗
PubMed1995

Cell cycle control of c-kit+IL-7R+ B precursor cells by two distinct signals derived from IL-7 receptor and c-kit in a fully defined medium.

An important goal for the investigation of the proliferation of mammalian cells is to establish a fully defined condition for culturing them in vitro. Here, we report establishment of a fully defined culture condition that supports the primary culture of normal c-kit+IL-7 receptor (IL-7R)+ B precursor cells without the aid of stromal cell lines. This defined culture condition contains IL-7, the ligand for c-kit, transferrin, insulin, and bovine serum albumin as protein components. By using the cell lines derived from RAG2(-/-) mice, which do not differentiate into c-kit- stage, we have evaluated the role of each protein in the cell cycle progression of c-kit+IL-7R+ B precursor cells. Since B precursor cells can grow without insulin, c-kit remains a sole functional receptor tyrosine kinase for their growth. While both c-kit ligand (KL) and IL-7 are the requisite molecules for sustained proliferation of B precursor cells, each molecule plays distinct roles. IL-7 starvation results in prompt arrest of the cells at G1. An accumulation of the cells in the mitotic phase was also detected. Thus, the major role of IL-7 is to regulate the G1/S transition and the process of cytokinesis of B precursor cells. Although prolonged KL starvation over 48 h resulted in accumulation of G1 cells, its effect could not be detected within 24 h, which is long enough for all the cells to complete one cell cycle. This suggests that KL might be involved in the cell cycle progression of B precursor cells in a manner that its signal could still be effective in the one or two cell cycles that follow. Although molecular nature of the signals underlying the present observation awaits future investigation, the method described in this report would provide a useful model system for investigating the signaling pathways that are involved in the cell cycle progression of B precursor cells.

Animals↗
PubMed1995

Extraordinary enhancement of the cleavage activity of a DNA-armed hammerhead ribozyme at elevated concentrations of Mg2+ ions.

As part of an ongoing effort to characterize structure-function relationships, activities of all-RNA and DNA-armed hammerhead ribozymes were examined. An analysis of the dependence on the concentration of Mg2+ ions of cleavage rates revealed that, whereas the kcat of the reaction catalyzed by the all-RNA ribozyme reached a maximum value of about 18 min-1 at a concentration of about 200 mM Mg2+ ions, that of the DNA-armed ribozyme increased linearly as the concentration of Mg2+ ions was increased above 300 mM, finally reaching a value of more than 100 min-1 at 700 mM Mg2+ ions. These results suggest that the potential activity of a hammerhead ribozyme might be greater than is usually recognized.

Base Sequence↗
PubMed1995

Generality of the NUX rule: kinetic analysis of the results of systematic mutations in the trinucleotide at the cleavage site of hammerhead ribozymes.

In order to study in detail the generality of the NUX (N = A, U, G, or C; X = A, U, or C) rule for the GUC triplet adjacent to the cleavage site in hammerhead ribozymes, two kinetic parameters, namely, kcat and Km, were determined for substrates with mutations in this triplet, which included double mutants with mutations of both N and X. All substrates with mutated cleavage sites were cleaved with reduced efficiency compared to the wild type. However, some mutations mainly affected kcat and others mainly affected Km, a phenomenon that could not have been predicted from previous results. A as the first or third base increased Km by 35- or 30-fold, respectively, while the effect on kcat was small. U as the first or third base decreased kcat by 8- or 15-fold, respectively, while the effect on Km was small. The effect of C as the first base on kinetic parameters was relatively small. The kinetic parameters of double mutants generally were determined by the effects of both individual point mutations. The AUA triplet gave a very much higher kcat than the other double mutants tested. In general, all of the mutants except for the mutant substrate with the CUC triplet had very low cleavage efficiencies, which ranged from 0.6% to 8% of the wild-type value, as a result of the deleterious effects of the mutations on kcat, Km, or both.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Identification of two transcripts of AML1/ETO-fused gene in t(8;21) leukemic cells and expression of wild-type ETO gene in hematopoietic cells.

The t(8;21) is a common chromosomal abnormality, preferentially associated with acute leukemia showing features of myeloid differentiation. Recently, two genes--AML1, which has a unique runt domain, and ETO (MTG8)--have been isolated from the chromosomal breakpoint. In this study, we isolated and identified two fused transcripts from a leukemic cell line carrying t(8;21). AML1 and ETO were fused at the same position in these transcripts. One of the transcripts codes a unique domain, including two zinc finger domains and three proline- and one leucine-rich region. The other transcript codes only for one proline- and leucine-rich region but lacks zinc finger domains. We demonstrated by polymerase chain reaction (PCR) analysis that 1) these two transcripts are consistently expressed in leukemic cells with t(8;21) obtained from patients and 2) expression of AML1 was not restricted to the particular stage of hematopoietic differentiation but was present in all hematopoietic cells investigated. We also provide evidence that two wild types of ETO transcripts containing the region of the ETO gene in fused transcripts are expressed in hematopoietic cells from different lineages. The widespread expression of AML1 and ETO in hematopoietic cells suggests a fundamental role of these proteins in hematopoiesis. Furthermore, the differences in the carboxy termini of ETO may modulate the activity of fused proteins resulting from the chromosomal translocation t(8;21).

Acute Disease↗
PubMed1995

Steel factor directs melanocyte development in vitro through selective regulation of the number of c-kit+ progenitors.

Studies of mice containing mutations in the genes for a receptor tyrosine kinase, c-kit, or its cognate ligand, Steel factor (SLF), establish that this signaling pathway is required for the development of melanocytes from their precursors in the embryonic neural crest (NC). In order to define the mechanism of this requirement, we have labeled cells expressing c-kit with an anti-c-kit antibody (ACK2) and studied the action of SLF on these cells in cultures of murine trunk NC. c-kit positive (c-kit+) cells first appeared after 2 days in culture and were morphologically indistinguishable from other NC cells. These cells subsequently expressed tyrosinase-related protein, an early marker for the melanocyte lineage, and became pigmented in the presence of a phorbol ester. Further, elimination of the c-kit+ population, by incubating the cultures in ACK2, resulted in the ablation of the melanocyte population, but had no effect on the generation of other neural crest derivatives. These data indicate that c-kit+ cells arising from the neural crest are melanocyte progenitors. The addition of SLF to these cultures stimulated an increase in the number of c-kit+ cells, and further studies indicated that SLF acts as both a survival and a proliferative factor for c-kit+ cells. These findings provide a mechanism of regulation of melanocyte development, whereby c-kit is exclusively expressed by melanocyte progenitors within the neural crest precursor population, and subsequent survival and proliferation of these progenitors is regulated by SLF.

Animals↗
PubMed1995

c-kit-dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract.

In vivo injection of a neutralizing, monoclonal antibody (ACK2) to the receptor tyrosine kinase (c-kit) disrupts the normal motility patterns of the mouse small intestine. Immunohistochemical studies showed that cells expressing c-kit-like immunoreactivity (c-kit-LI) decreased in numbers in response to ACK2, but the identity of these cells is unknown. We investigated the identity and development of the cells that express c-kit-LI in the mouse small intestine and colon. Cells in the region of the myenteric plexus and deep muscular plexus of the small intestine and in the subserosa, in the myenteric plexus region, within the circular and longitudinal muscle layers, and along the submucosal surface of the circular muscle in the colon were labeled with ACK2. The distribution of cells that express c-kit-LI was the same as that of interstitial cells (ICs). In whole-mount preparations cells with c-kit-LI were interconnected, forming a network similar to the network formed by cells that stained with methylene blue, which has been used as a marker for ICs in the mouse gastrointestinal tract. Immunocytochemistry verified that ICs were labeled with ACK2. Multiple injections of animals with ACK2 between days 0 and 8 post partum (pp) caused a dramatic reduction in the number of ICs compared to control animals. From an ultrastructural point of view, the proliferation and development appeared to be suppressed in some classes of ICs, while others displayed an altered course of development. Functional studies showed that the decrease in ICs was accompanied by a loss of electrical rhythmicity in the small intestine and reduced neural responses in the small bowel and colon. Morphological experiments showed that c-kit-positive cells are ICs, and physiological evidence reinforced the concept that ICs are involved in generation of rhythmicity and translation of neural inputs in gastrointestinal smooth muscles. Controlling the development of ICs provides a powerful new tool for the investigation of the physiological role of these cells.

Animals↗
PubMed1995

Making the in-vitro model closer to actual B lymphopoiesis in the bone marrow.

After more than a decade since Whitlock and Witte established an in-vitro long-term culture of bone marrow B lineage cells, c-kit+IL-7-receptor(R)+B precursor cells in fresh bone marrow are now able to be grown under a fully defined culture condition containing only BSA, transferrin, IL-7 and the ligand for c-kit(Kit-ligand;KL) as protein components. On the other hand, previous studies indicated that the actual intramarrow B-cell-genesis is a complex process involving multiple stromal cell-derived molecules. Thus, the next step for the culture of B-cell genesis is to develop this simple culture into a new defined culture of B-cell-genesis that is closer to the actual process. In this article, we will describe how this defined culture condition has developed from the original Whitlock-Witte type culture, how the B precursors under this culture-condition are different from that in the bone marrow, and finally our biased view on the future direction to which this defined culture should develop.

Animals↗
PubMed1995

Effects of monoclonal anti-c-kit antibody (ACK2) on melanocytes in newborn mice.

Previous studies indicate that c-Kit is required for postnatal melanocyte development. To understand the precise mechanisms of c-Kit dependence, we studied melanocyte development in newborn C57BL/6 mice by means of peritoneal injection of a monoclonal anti-c-Kit antibody (ACK2), which blocks c-Kit functions. The mice were injected once or more with ACK2 at various intervals after birth. In experiment 1, skin samples were examined on day 10 post-partum and in experiment 2 they were examined daily until day 10 post-partum. We studied melanocytes in the hair follicles, epidermis, and dermis by light and electron microscopy with dopa reactions and immunohistochemistry. Epidermal melanocytes in untreated mice were dopa negative and c-Kit positive on day 0 post-partum but became dopa positive soon thereafter. In ACK2-treated mice, the earlier the mice received ACK2 injections after birth, the fewer melanocytes they had, not only in the epidermis, but also in follicles. In these mice, melanocytes that had undergone apoptosis in the dermis and the follicles were detected ultrastructurally. Some appeared to have produced tyrosinase, because they had dopa-positive melanosomes. These results suggest that melanocytes in newborn mice are c-Kit dependent and undergo apoptosis when c-Kit receptors are blocked by ACK2 in the early days after birth. During this c-Kit-dependent period, melanocytes differentiate from dopa negative to positive and migrate from the epidermis to hair follicles.

Animals↗
PubMed1995

Activity of artificial mutant variants of human growth hormone changes in charged residues around 62-67.

Our previous work has shown that the amino acid residues around 62-67 located in the connecting loop between helix I and II of human growth hormone (hGH) are important in eliciting the differentiation of preadipose 3T3-F442A cells to adipocytes. In this study, we evaluated the role of the charged residues around 62-67 in receptor binding and biological activity. Eight artificial mutant variants of hGH were prepared in Escherichia coli by site-directed mutagenesis. Replacement of Arg64 with Tyr (R64Y variant) resulted in a significant loss of binding to the somatogenic receptors on 3T3-F442A cells, but retained full adipose conversion activity on these cells. Replacement of Arg64 with Glu (R64E) produced a considerable loss in receptor binding and a significant loss in biological activity. hGH variants in which either Glu65 or Glu66 was replaced with Asp (E65D and E66D) and with Gln (E65Q and E66Q) showed a slight loss in binding activity and retained almost a full adipogenic activity. An E65P variant (replacement of Glu65 with Pro) possessed the same binding activity as hGH, although it failed to induce full biological activity. The insertion of Ala between Asn63 and Arg64 (63NAR) caused a marked loss in both activities. These results indicate that the positively charged Arg64 is important for receptor binding and thereby in eliciting the biological activity of hGH, while negatively charged Glu65 and Glu66 are less important. In addition, our findings confirm that the conformation and size of the loop region around Arg64 is important for the adipose conversion activity of hGH.

Adipose Tissue↗
PubMed1995

Physiological concentrations of retinoic acid suppress the osteoblastic differentiation of fetal rat calvaria cells in vitro.

The effects of retinoic acid (RA) on osteoblastic differentiation and activity were studied in fetal rat calvaria cells cultured for up to 24 days. Fetal bovine serum used for the experiments was treated with an anion-exchange resin to remove endogenous RA. The depletion of RA in the treated serum was confirmed by high-performance liquid chromatography and tritiated RA tracing. Under the culture conditions employed, the continuous presence of RA for 14 days at 10(-9) mol/l or higher decreased both alkaline phosphatase (ALP) activity on day 12 and the number of bone nodules on day 14 in a dose-dependent manner. Short-term (24 h) exposure to RA at 10(-8) mol/l, which is a physiological concentration, decreased and increased the levels of ALP and osteopontin mRNA on day 6, respectively. Retinoic acid at 10(-8) mol/l also increased the level of osteocalcin mRNA on day 12. However, these effects were not obvious at later stages (days 18 and 24). At a high concentration (10(-6) mol/l), RA increased the level of osteopontin mRNA on day 6 and decreased the levels of ALP and osteocalcin mRNA irrespective of culture period. These results suggest that, at physiological concentrations, RA suppresses the differentiation of osteoprogenitor cells and regulates osteoblastic functions.

Alkaline Phosphatase↗
PubMed1995

Factors influencing nifedipine-induced gingival overgrowth in rats.

Factors such as age, the dose of nifedipine administered in the diet, serum drug level, duration of drug administration, and sex which may influence nifedipine-induced gingival overgrowth were examined in a rat model using 20-, 50-, and 90-days-old male and female rats. Oral administration of nifedipine (50 to 250 mg/kg diet) increased the serum level of the drug in a dose-dependent manner in both males and females. However, a higher serum level was required in females than males to attain the same degree of gingival overgrowth. The minimum dietary concentrations of the drug required to elicit gingival overgrowth in males and females were 150 and 100 mg/kg, respectively, which gave respective minimum serum levels of 800 and 1100 ng/ml. The degree of overgrowth depended on the serum concentration of the drug after it had reached the required minimum in male and female animals. Administration of nifedipine (250 mg/kg diet) for 20 days was enough to induce maximal overgrowth, but this induction occurred only in rats that started to receive the drug when they were 20 days old, not in those that started at 50 and 90 days of age for the same administration period of 55 days, and the overgrowth regressed and the gingiva were normal 40 days after ceasing drug administration. These results suggest that gingival overgrowth occurred in accordance with the drug concentration in the diet, as well as that in the serum, and was more likely to occur in males and younger individuals.

Age Factors↗
PubMed1995

Expression of AML1 and ETO Transcripts in hematopoietic cells.

Recently, two genes, AML1 and ETO have been isolated from the chromosomal breakpoint of t(8;21). In this study, we isolated and identified fusion transcripts from a leukemic cell line carrying t(8;21). We demonstrated by PCR analysis that these transcripts are consistently expressed in fresh leukemic cells with t(8;21). On the other hand, the wild type of ETO is expressed in several hematopoietic cells from different lineage, while the expression of AML1 was present in all hematopoietic cells investigated. These widespread expression suggests these molecules play an essential role in hematopoiesis.

Base Sequence↗
PubMed1994

cDNA sequence and structure of a gene encoding trout testis high-mobility-group-1 protein.

Perchloric acid extraction of trout testis nuclei revealed the presence of two large high-mobility-group (HMG) proteins, HMG-T1 and HMG-T2. The sequence of a complete cDNA (1407 bp) for trout testis HMG-1 protein (referred as to HMG-T1) has been determined. The deduced HMG-T1 protein contains 203 amino acids with more than 86% similarity to mammalian HMG-1 proteins. A single-sized mRNA for HMG-T1 has been detected by Northern-blot analysis consistent with the size derived from the HMG-T1 cDNA. Amplification of human and trout genomic DNAs by polymerase chain reaction using primers specific for trout and human HMG-1 cDNAs revealed that unlike the human genome, which contains predominantly intronless HMG-1 sequences, intronless HMG-T1 sequences were not found in the fish genome. Southern-blot analysis suggested that the trout testis HMG-1 gene is encoded by at least two sequences with high similarity. A gene encoding HMG-T1 protein has been isolated from a trout testis genomic library and by PCR of trout genomic DNA (3879 bp). The trout testis HMG-1 gene is organized into five exons (four exons corresponding to the protein-coding region) and its exon/intron boundaries are identical to those of the human HMG-2 gene [Shirakawa, H. & Yoshida, M. (1992). J. Biol. Chem. 267, 6641-6645] suggesting the evolution of HMG-1 and HMG-2 genes from a common ancestor.

Amino Acid Sequence↗
PubMed1994

Identification of phosphate oxygens that are important for self-cleavage activity of the HDV ribozyme by phosphorothioate substitution interference analysis.

A phosphorothioate substitution interference assay was used to investigate the role of the pro-Rp oxygens of phosphate groups in the self-cleavage reaction of the genomic human hepatitis delta virus (HDV) ribozyme. Incorporation of several different phosphorothioates (NTP alpha S) into the HDV ribozyme inhibited the self-cleavage activity. Incorporation of uridine 5' phosphorothioate or adenosine 5' phosphorothioate maintained 72% of the original self-cleavage activity whereas incorporation of guanosine 5' phosphorothioate or cytosine 5' phosphorothioate into the precursor reduced self-cleavage activity to about 20% in each case. Using partially substituted phosphorothioate-modified transcripts, we identified the pro-Rp oxygens that are important for the ribozyme activity, and they are located at positions 0, 1, 4, 5, 21, 24, 25, 27, 28, 30-34, 40, 43 and 75. In particular, the pro-Rp oxygens at positions 0, 1 and 21 are appear to be critical for the self-cleavage activity of the HDV ribozyme.

Base Sequence↗