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N Harada

Publications and source records attributed to N Harada.

At least 451 records · Page 25Linked to original sources
PubMed1992

Detection of the granulocyte colony-stimulating factor receptor using biotinylated granulocyte colony-stimulating factor: presence of granulocyte colony-stimulating factor receptor on CD34-positive hematopoietic progenitor cells.

Granulocyte colony-stimulating factor (G-CSF) was linked to NHS-biotin to yield biotinylated G-CSF (b-G-CSF), which retained the ability to stimulate colony formation by normal bone marrow (BM) cells in methylcellulose. The use of streptavidin-phycoerythrin conjugate in conjunction with flow cytometry demonstrated that the binding of biotinylated G-CSF to its receptor is saturable, competitive, and specific. A 100-fold molar excess of unlabeled G-CSF almost completely inhibited the binding of the biotinylated G-CSF to the human leukemia cell line U937, which is known to possess the G-CSF receptor. G-CSF receptors were clearly detected by flow cytometry on adult human peripheral granulocytes and monocytes, but not on lymphocytes. Using this method, the expression of G-CSF receptors on hematopoietic progenitor cells in bone marrow and umbilical cord blood, detected as CD34-positive (CD34+) cells, were examined. A small but significant number of CD34+ cells were detected among the bone marrow mononuclear cells and umbilical-cord-blood mononuclear cells (4.28% +/- 0.31%, 1.09% +/- 0.20%, respectively). The percentage of CD34+ BM mononuclear cells was significantly higher than for cord blood mononuclear cells (P less than 0.01). These CD34+ cells were then analyzed by biotinylated G-CSF binding. CD34+ cells from bone marrow contained 25.8% +/- 7.9% G-CSF receptor positive cells and those from cord blood possessed 29.2% +/- 7.0% of G-CSF receptor-positive cells. The difference was not statistically significant.

Antigens, CD↗
PubMed1992

Direct contractile effect of motilin on isolated smooth muscle cells of guinea pig small intestine.

We examined the direct effect of motilin on longitudinal and circular smooth muscle cells isolated from the guinea pig small intestine. In addition, the effects of 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride (TMB-8, an inhibitor of intracellular Ca(2+)-release), verapamil (a voltage-dependent Ca(2+)-channel blocker), and removal of extracellular Ca2+ were investigated to evaluate the role of intracellular Ca2+ stores and extracellular Ca2+ on the muscle contraction induced by motilin. The effects of atropine (a muscarinic receptor antagonist), spantide (a substance P receptor antagonist) and loxiglumide (a CCK-receptor antagonist) were also examined to determine whether the motilin-induced contraction was independent of those receptors. Motilin induced a contraction of the longitudinal and circular smooth muscle cells in a dose-dependent manner with the maximal effect attained after 30 seconds of incubation. The ED50 values were 0.3 nM and 0.05 nM, respectively. TMB-8 suppressed completely the motilin-induced contraction of both types of smooth muscle cells. Verapamil had only a slight suppressive effect. Removal of extracellular Ca2+ did not have any significant influence on motilin-induced contraction. The contractile response to motilin was not affected by atropine, spantide or loxiglumide. Our findings showed that:1) motilin has a direct contractile effect on both longitudinal and circular smooth muscle cells; 2) this contractile effect is not evoked via muscarinic, substance P or CCK receptors, and 3) the intracellular release of Ca2+ plays an important role in the contractile response to motilin on both types of smooth muscle cells.

Animals↗
PubMed1992

Aromatase as a cellular marker of testosterone action in the preoptic area.

We recently showed, using a new immunocytochemical technique, that aromatase-immunoreactive neurons are a specific marker for the sexually dimorphic medial preoptic nucleus (POM) in quail and that the number of these immunoreactive cells is markedly increased by a systemic treatment with testosterone (T). Since the POM is a key site for the activation of copulatory behavior by T and this androgen must be converted into estrogen by local aromatization within the POM before it can exert its behavioral effects, we used aromatase immunocytochemistry to map, at a cellular level of resolution, the areas that are destroyed by electrolytic lesions or that are stimulated by the stereotaxic implantation of T in the preoptic area (POA). These measures of the cellular action of T in the preoptic area were then correlated with the behavior of the animals to identify the parts of the POA that are critical in the activation of behavior. The electrolytic lesions of the POA disrupted the activation of male sexual behavior by T only if they destroyed a significant part of the POM. All lesions reduced the volume of the dimorphic nucleus and the absolute number of its aromatase-immunoreactive neurons, but the density of these cells in the remaining POM was not affected, suggesting that the volume change in the nucleus reflected a centripetal displacement of its boundaries rather than an overall shrinkage of the structure. Stereotaxic T implants in or close to POM activated male copulatory behavior and increased the volume of the POM and the number of its aromatase-immunoreactive cells. These neuroanatomical effects were more prominent on the side of the implant, but they were also detected on the contralateral side. Correlative analyses suggested that a part of the POM just rostral to the anterior commissure is critical for the activation of copulatory behavior. The best correlations between the behavioral deficits induced by electrolytic lesions and the size of the lesions were indeed observed in this area. In addition, high correlations were also observed between the behavior activated by T implants and the POM size or number of aromatase-immunoreactive cells that were induced by T in this area. Aromatase immunocytochemistry therefore appears as a useful tool to map the brain areas in which T action is presumably critical for the activation of male sexual behavior. It has allowed us to identify in the present studies a small part of the sexually dimorphic POM that is closely associated with behavior.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Induction of phosphatidylinositol-glycan-linked Fc gamma RIII in human monocytic THP-1 cells by transforming growth factor-beta 1 and retinoic acid.

Human monocytic leukemic cell line THP-1 was incubated with transforming growth factor-beta 1 (TGF-beta 1) and retinoic acid (RA) and the expression of Fc gamma RIII was investigated. Fc gamma RIII was induced after incubation of the cells with both TGF-beta 1 and RA but not with either TGF-beta 1 or RA alone. Such effects of TGF-beta 1 and RA were not detected on human promyelocytic HL-60 cells. Northern blot analysis revealed the induction of Fc gamma RIII transcripts in THP-1 cells. Furthermore, the Fc gamma RIIIs newly expressed on the cell surface were cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC) and reacted with monoclonal antibody MG38 which specifically binds to granulocyte-type Fc gamma receptors. These results indicated that TGF-beta 1 could induce phosphatidylinositol-glycan-linked Fc gamma RIII (Fc gamma RIII-I) in THP-1 cells in the presence of RA.

Antigens, Differentiation↗
PubMed1992

Regulation of aromatase cytochrome P-450 (estrogen synthetase) transcripts in the quail brain by testosterone.

The aromatase cytochrome P-450 (P-450AROM) cDNA, which was identified by homologies in the DNA and in the deduced amino acid sequences with human P-450AROM cDNA, was isolated from a brain cDNA library of Japanese quail, demonstrating the presence of RNA transcripts of P-450AROM in the quail brain. To determine trace amounts of P-450AROM mRNA in the brain and to examine the effects of testosterone on its expression, a quantitative PCR method of RNA transcripts was developed. Brain total RNA was subjected to reverse transcription reaction and then quantitated by PCR from cDNA with a fluorescent dye-labeled primer. The quantity of P-450AROM mRNA was calculated by using an internal standard of modified P-450AROM (m-P-450AROM) RNA. The brain P-450AROM was primarily transcribed in the hypothalamus area (1.15 +/- 0.14 amol/micrograms of RNA) and traces of transcripts only were detected in the cerebellum (0.038 +/- 0.005 amol/micrograms of RNA). The P-450AROM mRNA in the hypothalamus of castrated quail was low (0.270 +/- 0.078 amol/micrograms of RNA) and increased 4- to 5-fold following treatment with testosterone. These results demonstrate, for the first time, that the increase in P-450AROM activity that is observed in the brain following treatment with testosterone results from a pretranslational regulation of the P-450AROM by androgens.

Androgens↗
PubMed1992

Serum granulocyte colony-stimulating factor levels in umbilical cord blood of normal full-term neonates.

We measured granulocyte colony-stimulating factor (G-CSF) levels in cord blood of 59 normal full-term neonates immediately after birth and the subsequent changes in G-CSF levels of 16 cases by our modified enzyme-linked immunosorbent assay (ELISA) for G-CSF. Ten out of 59 cases examined (17%) showed G-CSF levels in cord blood after delivery between 20 and 57 pg/ml, although in the remaining cases, the G-CSF levels were below 20 pg/ml, which was our minimal detection level. A direct relationship between G-CSF levels and white blood cell count, absolute granulocyte numbers in cord blood, gestation age or weight was not observed. Although G-CSF levels in cord blood after delivery remained below 20 pg/ml in 5 cases out of 16 tested, those in the remaining 11 cases (69%) subsequently became elevated after delivery. The peak G-CSF level in cord blood after delivery ranged from 26-364 pg/ml, and the time of it was between 4.5 and 18 h. As G-CSF level per wet weight of placenta was high (124 x 29 pg/ml), these subsequent elevations of G-CSF in cord blood after delivery may result from a gradual influx from the placenta.

Enzyme-Linked Immunosorbent Assay↗
PubMed1992

Microdissection of human chromosomal regions 8q23.3-q24.11 and 2q33-qter: construction of DNA libraries and isolation of their clones.

Human chromosomal regions 8q23.3-q24.11 and 2q33-qter were microdissected, DNAs from the regions were amplified with the primer-linker method of polymerase chain reaction (PCR), and their DNA libraries were constructed by cloning into pUC19. The primer-linker PCR involved Sau3AI digestion of microdissected chromosomal DNAs, ligation of the digests to a 10mer DNA linker and 24mer primer, filling the recessed 3' ends, and PCR amplification using the 24mer DNA as a primer. A total of 3.5 x 10(4) pUC19 recombinants (8q library) from the 8q region and 5.0 x 10(4) pUC clones (2q library) from the 2q region were obtained. From the 8q library, 60 pUC clones were selected, while 88 pUC-clones were selected from the 2q library. These clones were Southern blot analyzed on hybrid cell panels with or without human chromosome 8 or 2. Twelve (20%) of the 60 8q-derived clones were unique DNA sequences, and 9 were subjected to deletion analysis in the genomic DNA of two patients, one with trichorhino-phalangeal syndrome (TRPS) type I and the other with TRPS type II, both with del(8) (q23.3q24.13). Five of the 9 pUC clones tested showed a one-copy density in both patients, an indication that the clones map to the region deleted in both patients. Screening a genomic DNA library constructed in the phage revealed a clone with a 9.4-kb insert and a one-copy density in both patients. From the 2q library, 15 (17%) of the 88 pUC clones obtained were unique sequences. When a phage library was screened, 8 clones were obtained: 4 were identical and 2 were overlapping sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities, Multiple↗
PubMed1992

A simple and efficient amplification method of DNA with unknown sequences and its application to microdissection/microcloning.

An alternative method for amplification of DNA with unknown sequences was developed. This involves the direct ligation of a primer oligodeoxyribonucleotide itself to restricted DNA fragments with unknown sequences to be amplified by PCR. The oligonucleotide need not be phosphorylated and need not be annealed with its complementary oligonucleotide in advance for ligation. The ligation reaction seems to be independent of the concentration of unknown DNA, proceeds in short time, and is efficient. The ligation efficiency was more than 30% at a low concentration, 10 fg/microliters, of DNA. This method was applied to a microdissection/microcloning of the short arm of human chromosome 2. Of 65 clones screened for the highly repetitive sequences with total human genomic DNA, eleven (17%) were positive. Their inserts ranged in size from 150 to 1,200 bp (average, 460 bp). In Southern blot analysis, thirty consecutive clones all detected signals common to both total human genomic DNA and mouse-human hybrid cell DNA containing only chromosome 2 of human origin. Among them, 24 (80%) were unique sequences, and 6 (20%) were multi-copy (or intermediate-repeat) sequences. Thus, this method is simple and efficient, and provides an alternative way to amplify unknown DNA.

Base Sequence↗
PubMed1992

Regulation of myeloid-specific calcium binding protein synthesis by cytosolic protein kinase C.

Two calcium binding proteins, MRP-8 and MRP-14, are specifically synthesized in human myeloid cells. This paper shows that Me2SO, all-trans-retinoic acid (RA) and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3), but not 12-O-tetradecanoyl phorbol-13-acetate (PMA) are potent inducers of MRP-8/14 protein complex in human leukemic cells. Transforming growth factor-beta 1 (TGF-beta 1) is shown to enhance the inductive effect of RA and 1 alpha,25(OH)2D3. We have examined the possibility that MRP expression is regulated through the protein kinase pathway. Both cytosolic and membrane-bound protein kinase C (PKC) activities increased during differentiation by RA and 1 alpha,25(OH)2D3. PMA-treatment led to a decrease of cytosolic PKC activity and an increase of membrane-bound PKC activity in the presence of these differentiation inducers, while PMA alone resulted in low cytosolic and high membrane-bound PKC activities. PKC inhibitor H7 inhibited MRP synthesis in HL-60 cells treated with RA and 1 alpha,25(OH)2D3. These results suggest that cytosolic PKC activity may be involved in a stimulatory pathway of MRP synthesis and that protein phosphorylation reactions may play important roles in MRP expression during myelocytic differentiation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Cytokine receptors and signal transduction.

Cytokines play a vital role in coordinating immune and inflammatory responses. Unlike growth factor receptors with a tyrosine kinase, cytokine receptors have no intrinsic tyrosine kinase activity. Based on their structure, cytokine receptors are classified into several groups. High affinity receptors for IL-2, IL-3, IL-5, IL-6, and GM-CSF are composed of at least two distinct subunits, alpha and beta. The alpha subunits are primary cytokine binding proteins, and the beta subunits are required for formation of high affinity binding sites as well as for signal transduction. The GM-CSF, IL-3, and IL-5 receptors appear to share the same beta subunit in human, and therefore cross-talk among these cytokines may occur at the receptor level. High affinity receptors presumably are linked to various signal transduction pathways that lead to different cytokine functions. Differential expression of the cytokine receptors as well as reorganization of intracellular signalling pathways are critical for development of hemopoietic cells.

Amino Acid Sequence↗
PubMed1992

EndothelinB receptor on guinea pig small intestinal smooth muscle cells.

We investigated the binding characteristics of the endothelin (ET) receptor and the mechanism by which ET induces contraction of longitudinal smooth muscle cells of the guinea pig small intestine by using vasoactive intestinal contractor (VIC), a mouse variant of ET-2. A functional receptor for VIC was found to exist on longitudinal smooth muscle cells. These cells showed a similar binding of and contractile response to ET-1, ET-2, and ET-3. Inhibitors of both intracellular and extracellular Ca2+ movement attenuated the VIC-induced contraction of longitudinal smooth muscle cells. These results suggest that smooth muscle cells of the guinea pig small intestine express the ETB receptor that primarily mediates the contractile effect on smooth muscle cells. In addition, ET-induced contraction depends on intracellular as well as extracellular Ca2+.

Animals↗
PubMed1992

Ontogeny of aromatase messenger ribonucleic acid in mouse brain: fluorometrical quantitation by polymerase chain reaction.

A sensitive method was developed for the determination of trace amounts of aromatase mRNA in various tissues. Aromatase mRNA was quantitated by subjecting it to reverse transcription in the presence of an internal standard RNA and then amplifying the resulting cDNA by polymerase chain reaction with a fluorescent primer. The tissue distribution of aromatase mRNA in mice was examined by this polymerase chain reaction method. Results showed that aromatase mRNA is expressed in the brain, testis, and ovary, but scarcely at all in other tissues, including the placenta. In mouse brain, aromatase is mainly located in the forebrain, especially the diencephalon. In adult male and female diencephala, aromatase mRNA levels were 0.022 +/- 0.004 and 0.014 +/- 0.003 attomoles/micrograms total RNA, respectively. Aromatase in the diencephalon is known to participate in brain differentiation and sexual behavior, so changes in its mRNA levels in the brain during development were examined. Aromatase mRNA was first detected in the 12-day-old fetus and was found to increase rapidly during the fetal and neonatal periods. Its mRNA levels in male and female brains reached maxima of 0.068 +/- 0.008 and 0.059 +/- 0.006 attomoles/micrograms total RNA, respectively, 3-4 days after birth and then gradually decreased to adult levels. These observations are consistent with previous data indicating that the marginal period of neonatal imprinting of sexual differences is within a week after birth and suggest that brain aromatase may be important in sexual differentiation and behavior.

Animals↗
PubMed1992

Sibling cases of primary biliary cirrhosis associated with polymyositis, vasculitis and Hashimoto's thyroiditis.

Primary biliary cirrhosis (PBC) was diagnosed in two siblings, a 42-year-old male and his 49-year-old sister. Furthermore, they were complicated with three types of autoimmune diseases: polymyositis, Hashimoto's thyroiditis and vasculitis of the right axillary artery. Although PBC is often associated with other autoimmune diseases, reports of the association with polymyositis and vasculitis syndrome are rare. Familial clustering of PBC with autoimmune diseases is also rare.

Adult↗
PubMed1992

Intracellular free calcium concentrations in single taste receptor cells in the guinea pig.

Single, viable taste receptor cells were isolated from the tongue of the guinea pig by enzymatic digestion and mechanical dissociation. The cells could be classified into flask, spindle and intermediate shapes. The intracellular free calcium ion concentrations [(Ca2+)i] of these cells were determined using the Ca2+ sensitive dye fura-2 and digital imaging microscopy. All types of cells produced an irreversible increase in (Ca2+)i upon addition of Ca2+ ionophore ionomycin (1 microM) and denatonium (10 microM). There was no evidence of any increase in (Ca2+)i in the taste receptor cells in nominally Ca2+ free solution, and when stimulated by denatonium (10 microM). When 3 mM CaCl2 was added, the (Ca2+)i remarkably increased. This would suggest that the (Ca2+)i increase in the presence of denatonium mainly depended on calcium influx from the extracellular space. There was no increase in case of high potassium (50 mM and 150 mM) or saccharose (1 mM and 5 mM) stimulation. The hypothesis that the increase in (Ca2+)i controls biochemical mechanisms related to the bitter taste transduction process is worthy of further study.

Animals↗
PubMed1992

Responses of the solitary olfactory receptor cell of the guinea pig.

Olfactory receptor cells were isolated from the nasal mucosa of the guinea pig, using enzymatic digestion and mechanical dissociation. Under a phase-contrast microscope, the cells can be readily distinguished from other tissues, because of morphological characteristics. The intracellular free calcium ion concentrations ([Ca2+]i) of these cells were determined using the Ca2+ sensitive dye fura-2 and digital imaging microscopy. In the presence of 1 microM ionomycin, calcium-ionophore, there was an irreversible increase in [Ca2+]i in all the cells. During prolonged stimulation by 10 mM n-Amyl acetate under standard conditions, 16 of the 40 isolated olfactory cells responded with [Ca2+]i. There was no evidence of any increase in [Ca2+]i in the olfactory receptor cells in nominally Ca2+ free solution and stimulated by amyl acetate. When 3 mM CaCl2 was added, the [Ca2+]i increased remarkably. When cells were stimulated with amyl acetate in a CaCl2-depleted extracellular fluid supplemented with 10 microM verapamil (an L-type voltage-dependent calcium channel antagonist), no change occurred in [Ca2+]i. The addition of CaCl2 to this fluid did not elevate [Ca2+]i, in any cell. This would suggest that the [Ca2+]i increase in the presence of amyl acetate may mainly depend on the influx of calcium from the extracellular space. Our finding that the influx of extracellular calcium into cells was suppressed by verapamil suggests that the calcium channel, which is opened by amyl acetate, is a voltage-dependent L-type Ca2+ channel.

Animals↗
PubMed1992

The murine interleukin-4 receptor gene: genomic structure, expression and potential for alternative splicing.

The structure of the murine interleukin-4 receptor (mIL-4R) gene has been determined. The gene spans approximately 25 kilobases (kb) of DNA and is composed of 12 exons interrupted by 11 introns. The gene contains sequences accounting for all the sequences present in the functional mIL-4R cDNAs, including several exons which can encode DNA inserts found in recently cloned IL-4R cDNA variants. Thus expression of the gene may be regulated, at least in part, by alternative splicing. A combination of S1 nuclease protection and primer extension assays was used to localize the 5' end of the gene and demonstrated the use of multiple transcription initiation sites within this region. We have found that the overall intron-exon organization of the murine IL-4R gene is markedly similar to that of the murine erythropoietin receptor (m-epoR) and the human interleukin-2 receptor beta chain (hIL-2R beta), as well as the human growth hormone receptor (hGhR). This is consistent with the recent grouping of these receptors, on the basis of protein sequence homology, into the hematopoietin receptor gene superfamily. Such homology at the levels of both protein and gene structure suggest a divergent evolution of the receptors from a single primordial gene.

Animals↗
PubMed1992

Correlation between plasma alpha-cysteine proteinase inhibitor level and pyruvate kinase activity in vitamin E-deficient rats.

Weaning rats were divided into two groups, one group being fed a vitamin E-deficient diet, and the other an alpha-tocopherol-containing (50 mg/kg) control diet. Rats were killed at 1, 2, 3 and 4 months of feeding. The following results were obtained. 1. Both plasma and liver alpha-tocopherol levels decreased greatly by feeding vitamin E-deficient diet for 1 month, and thereafter decreased gradually by continued feeding. 2. Somehow different results were obtained concerning liver peroxidation value by the method of analysis. In the case of chemiluminescence, the value increased by vitamin E deficiency during the first 2 months, but thereafter, the value was almost unchanged. On the contrary, in the case of TBA-RS, the value increased gradually throughout the entire 4 months of feeding period. 3. Both plasma alpha-CPI level and pyruvate kinase activity increased by vitamin E deficiency, showing similar pattern of change with feeding period. Especially, marked increase of these values was observed in vitamin E-deficient rats fed for longer than 2 months, and differences from control groups were highly significant (in both cases, at 2 months, p < 0.005, and at 3 and 4 months, p < 0.001). And, in vitamin E-deficient group, including all the rats fed on test diet for 1 to 4 months, correlation between both values was very high, and was highly significant (r = 0.9060, p < 0.001).

Animals↗