Immunocytochemical study of the ultrastructure of peroxisomes and the effects of peroxisome proliferators.
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Animals↗
Biomedical subjects
M Terasawa
Publications and source records attributed to M Terasawa.
Effect of Y-24180, a platelet-activating factor-receptor antagonist, on the antigen-induced airway microvascular leakage in guinea pigs.
The inhibitory effect of Y-24180 ((+/-)-4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6, 9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine) on platelet-activating factor (PAF)- or antigen-induced airway microvascular leakage was studied in guinea pigs by oral administration. The tissue content of extravasated Evans blue dye was used as an index of plasma exudation in the trachea, main bronchi, central intrapulmonary airways and peripheral intrapulmonary airways. In all of these tissues, Y-24180 potently inhibited the leakage induced by PAF. The ED50 value of Y-24180 determined in each of the tissues was approximately 0.02 mg/kg, demonstrating that the inhibitory potency of Y-24180 is 4-6 times that of WEB 2086, another PAF antagonist. Even at a dose of 10 mg/kg, however, Y-24180 showed no inhibitory effect on the leakage induced by leukotriene (LT) D4, histamine or bradykinin. In the antigen-induced model of guinea pigs sensitized with aerosolized ovalbumin, Y-24180 (0.1-10 mg/kg) and WEB 2086 (1-100 mg/kg) potently inhibited the microvascular leakage in all of the examined airway tissues except for the trachea. At 1-100 mg/kg, however, both ONO-1078, an LT-receptor antagonist, and OKY-046, a thromboxane A2 synthetase inhibitor, prevented partially but not significantly the antigen-induced leakage. These results provide evidence that endogenous PAF partially mediates the antigen-induced airway microvascular leakage in guinea pigs.
Animals↗
[Functions of recombination proteins in meiosis].
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Animals↗
Localization of RecA-like recombination proteins on chromosomes of the lily at various meiotic stages.
The Rad51 and Lim15 proteins of lily, which are homologs of the bacterial RecA protein, were found on chromosomes in various stages of meiotic prophase 1. The presence of both Rad51 and Lim15 proteins as discrete foci on leptotene and zygotene chromosomes and their colocalization suggest that meiotic recombination begins at the leptotene stage with the cooperation of these proteins and continues in zygotene. Localization of the foci on or adjacent to the chromosomes suggests that these proteins bind to the chromatin loops that extend from the axial cores. The proteins in these foci may participate in the searching and pairing of homologous DNA sequences, as the RecA protein does. The different pattern of localization of the Rad51 protein between the leptotene and pachytene stages and the absence of the Lim15 protein in the pachytene stage suggest that the Rad51 protein plays different roles in these stages.
Amino Acid Sequence↗
[Mechanism of analgesic action of Y-23023, a new non-steroidal anti-inflammatory drug].
The analgesic mechanism of Y-23023, a new non-steroidal anti-inflammatory drug, was investigated in the writhing response induced by intraperitoneal injection of kaolin and captopril in mice. Y-23023 (0.1-1 mg/kg, p.o.) suppressed the writhing frequency in a dose-dependent manner. Y-23023 also significantly reduced the increased levels of prostaglandin (PG) and bradykinin (BK) in the peritoneal cavity. In contrast, indomethacin, diclofenac sodium, loxoprofen sodium and mefenamic acid inhibited the writhing response, but their efficacies were lower than that of Y-23023. The peritoneal PG levels were dose-dependently reduced to the same extent as Y-23023, whereas the BK levels were not. M1, an active metabolite of Y-23023, inhibited the cyclooxygenase from sheep vesicular gland in a concentration-dependent manner, and its potency was similar to that of indomethacin. These results suggest that in addition to the suppressive effect on PG production via inhibition of cyclooxygenase, the inhibitory effect on BK production is involved in the analgesic action of Y-23023, unlike indomethacin and diclofenac sodium.
Analgesics↗
[Analgesic and anti-inflammatory effect of (+/-)-N,N- dimethylcarbamoylmethyl 2-[7-(2-methyl-5H-[1] benzopyrano [2,3-b] pyridyl)] propionate (Y-23023), a new non-steroidal anti-inflammatory drug].
The analgesic and anti-inflammatory activities of Y-23023, a new nonsteroidal analgesic and anti-inflammatory compound, were investigated in acute, subacute and chronic pain models in rats. In the carrageenin paw edema test, Y-23023 (0.3-3 mg/kg, p.o.) dose-dependently inhibited hyperalgesia assessed by the Randall-Selitto method and paw edema. Y-23023 (1 mg/kg, p.o.), when administered at 2 hr after carrageenin injection, significantly elevated the reduced pain threshold without affecting paw edema. Therefore, the antinociception activity induced by Y-23023 was not the result of its anti-inflammatory activity. In addition, Y-23023-induced antinociception was resistant to post-treatment with naloxone (2 mg/kg, s.c.), but was antagonized by intraplantar injection of prostaglandin E2 (1 microgram/paw). These results suggest that Y-23023 produces a peripheral analgesic effect mediated by inhibition of prostaglandin production. Y-23023 (0.3-10 mg/kg, p.o.) also had a potent inhibitory effect on the silver nitrate-induced arthritic pain. In suppressing acute and subacute pain, Y-23023 was more potent than diclofenac sodium, indomethacin and loxoprofen sodium. The analgesic and anti-inflammatory activities of Y-23023 (0.1-1 mg/kg/day, p.o.) on the adjuvant-induced hyperalgesia and the paw swelling were nearly equipotent to diclofenac sodium and indomethacin, but were more potent than loxoprofen sodium. Therefore, Y-23023 would be regarded to show predominantly strong analgesic activity in acute and subacute pain when compared with reference drugs. The above results suggest that Y-23023 is a novel analgesic compound with an anti-inflammatory activity in the clinical field.
Animals↗
Ca(2+)-calmodulin mediated modulation of the electrical coupling of ventricular myocytes isolated from guinea pig heart.
The mechanism for the regulation of junctional conductance (gj) by Ca2+ was examined in paired ventricular myocytes isolated from guinea pig heart. One cell of the pair was voltage-clamped by a single-patch pipette, and gj was measured after perforation of the non-junctional membrane of the partner cell. The average value of gj under control condition at pCa 9.0 was 181 +/- 13 nS (n = 30). An elevation of calcium concentration ([Ca2+]i) in the intracellular perfusate from pCa 9.0 to pCa 5.7 resulted in a decrease of 32 +/- 5% in gj (n = 18). In myocytes pretreated with W7 (10(-4) M), a similar elevation of [Ca2+]i caused a decrease of only 10 +/- 3% in gj (n = 6), indicating a protective action of W7 against Ca(2+)-mediated electrical uncoupling. W5 (10(-4) M), a non-chlorinated derivative of W7, did not show such a protective action. Calmodulin (10(-5) M) had no effects on gj at pCa 9.0. However, at moderately elevated [Ca2+]i condition at pCa 7.0, calmodulin (10(-5) M) decreased gj by 29 +/- 6% (n = 4). These results suggest that calmodulin may play an important role in the Ca(2+)-mediated regulation of gap junctional channel function in the cardiac ventricular cells.
Action Potentials↗
A newly synthesized bifunctional inhibitor, W-77, enhances adriamycin activity against human ovarian carcinoma cells.
A newly synthesized calmodulin antagonist, (S)-P-(2-aminoethyloxy)-N-[2-(4-benzyloxy-carbonylpiperazinyl++ +)-1-(P-methoxybenzyl)ethyl]-N-methylbenzenesulfonamide dihydrochloride (W-77), acts as a calcium-independent uncompetitive antagonist which binds to glutathione-S-transferase (GST). We purified GST from human placenta using drug affinity chromatography on a column of W-77 coupled with Sepharose 6B and demonstrated that W-77 bound to GST. A spectrophotometric assay also showed that W-77 inhibited GST activity. We prepared Adriamycin-resistant and -sensitive cells from human ovarian serous cystadenocarcinomas. Immunoblot analysis revealed that GST expression was increased in the Adriamycin-resistant cells. We also purified GST from Adriamycin-resistant cells and found that W-77 bound to the GST obtained from these ovarian carcinoma cells. Adriamycin resistance was partially overcome by the addition of W-77 (10 microM) to the cultured cells. In addition, we investigated the effect of W-77 on P-glycoprotein. Northern blot analysis revealed MDR1 gene expression in Adriamycin-resistant cells. Although W-77 was less potent in increasing the intracellular Adriamycin content than verapamil, it was more effective in overcoming Adriamycin resistance. These results suggest that W-77 enhances the antitumor activity of Adriamycin by inhibiting both GST and P-glycoprotein.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Distinct regional localization of neurocalcin, a Ca(2+)-binding protein, in the bovine adrenal gland.
Neurocalcin (molecular weight 23,000 and 24,000) is a Ca(2+)-binding protein with three putative Ca(2+)-binding domains and is present in large amounts in nervous tissues. Neurocalcin isoproteins separated by C18 reverse-phase column chromatography are insoluble in buffer solution and it is impossible to determine the dissociation constant of neurocalcin with Ca2+. To overcome this difficulty, recombinant neurocalcin was synthesized, based on one of the cDNAs of the neurocalcin isoproteins. Stoichiometric titration experiments, using recombinant neurocalcin, indicated that this protein bound 2 mol Ca2+/mol protein and that the apparent dissociation constant for Ca2+ was 2.2 mumol/l, suggesting that neurocalcin plays a physiological role in cellular function. Immunoblotting showed that neurocalcin is present in the bovine adrenal gland in addition to the nervous tissues. Neurocalcin, identified by immunoblotting, was purified from the bovine adrenal gland. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of neurocalcin from the bovine brain showed 23 kDa and 24 kDa double bands, while SDS-PAGE of neurocalcin from the adrenal gland showed a single band of apparently 24 kDa, suggesting that the expression of neurocalcin isoproteins differs from tissue to tissue. The content of neurocalcin in the adrenal gland was 10 micrograms protein/100 g wet tissue. Immunohistochemical analysis showed the occurrence of neurocalcin in zona glomerulosa and adrenal medulla but not in zona fasciculata or zona reticularis. The restricted localization of neurocalcin in the adrenal gland suggests that a similar Ca2+signal pathway may be present in zona glomerulosa a nd the adrenal medulla.
Adrenal Glands↗
Industrial production of biochemicals by native immobilization.
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Aspartic Acid↗
[Advance in eosinophil chemotaxis assay method--automatic count of migrated cells by the image analyzing system in the Boyden method].
Eosinophils, which are prominent inflammatory cells in the asthmatic airway, are attracted to the airway by several chemoattractants. At present, eosinophil chemotaxis is studied employing the Boyden Millipore chamber system in vitro. In the Boyden method, the number of migrated cells are evaluated by direct microscopic observation of individual cells. This microscopic procedure usually requires much time and it is difficult to obtain objective results. Therefore, we have developed a new fractional measurement technique, using the image analyzing system connected with a color video camera and a computer for counting the eosinophils which have migrated into a filter. Using this system with the Boyden method, migrated cells were observed more objectively and quickly. Therefore, this image analyzing system with the Boyden method, may be a useful technique for the fractional measurement of migrated eosinophils in a sample mixture of eosinophils and neutrophils.
Cell Movement↗
Calcyclin and calvasculin exist in human platelets.
Using Ca(2+)-dependent affinity chromatography on a synthetic compound (W-7)-coupled Sepharose column, three distinct Ca(2+)-binding proteins have been identified in human platelets. The molecular mass of these three distinct proteins was estimated to be 10, 10.5, 17 kDa, respectively, by polyacrylamide gel electrophoresis in the presence of SDS. The partial amino acid sequence revealed these proteins have EF-hand structures and high homology to the predicted proteins, calcyclin, calvasculin, and calmodulin. Calcyclin and calvasculin have been considered as probably having roles in the control of cell proliferation, but the existence of these two proteins in platelets suggests that they have other intracellular functions related to the Ca(2+)-signal transduction system.
Amino Acid Sequence↗
Neurocalcin: a novel calcium-binding protein from bovine brain.
A novel calcium-binding protein (molecular weight 23,000-24,000, pI 5.3-5.5), which we term neurocalcin, was identified in bovine brain. Using calcium-dependent drug affinity chromatography ((S)-P-(2-aminoethyloxy)-N-[2-(4-benzyloxycarbonylpiperazinyl++ +)-1-(P- methoxybenzyl)ethyl]-N-methylbenzene-sulfonamide dihydrochloride, W-77, -coupled Sepharose 6B), we purified neurocalcin from bovine brain. The partial amino acid sequence of neurocalcin revealed it to be an as yet unidentified protein with three putative calcium binding sites (EF-hands). Further purification and sequence analysis demonstrated the presence of four isoprotein forms designated alpha, beta, gamma 1, and gamma 2. When the 165 sequenced residues of neurocalcin beta are compared with sequences of other proteins, neurocalcin beta has a 38.2% sequence homology with visinin and 45.5% with recoverin (Yamagata, K., Goto, K., Kuo, C.-H., Kondo, H., and Miki, N. (1990) Neuron 2, 469-476; Dizhoor, A. M., Ray, S., Kumar, S., Niemi, G., Spencer, M., Brolley, D., Walsh, K. A., Philipov, P. P., Hurley, J. B., and Stryer, L. (1991) Science 251, 915-918). Both visinin and recoverin are expressed specifically in retinal photoreceptors and are not found in brain. Unlike visinin and recoverin, neurocalcin is purified not only from retina but also from bovine brain. Our results suggest that neurocalcin is a recoverin-like protein expressed in bovine brain.
Amino Acid Sequence↗
Neurocalcin, a novel calcium binding protein with three EF-hand domains, expressed in retinal amacrine cells and ganglion cells.
Neurocalcin (molecular weight 23,000 and 24,000) is a newly identified Ca2+ binding protein with three EF-hand domains and has a strong amino acid sequence homology with visinin and recoverin (Terasawa, M., Nakano, A., Kobayashi, R., and Hidaka, H. J. Biol. Chem. In press). We produced antibody against neurocalcin. Immunoblotting showed the presence of neurocalcin in bovine retina as well as brain, suggesting that neurocalcin was a neuron specific Ca2+ binding protein. Immunohistochemistry revealed the expression of neurocalcin in retinal amacrine cells and ganglion cells but not in the photoreceptor layer. This distribution of neurocalcin was quite different from that of visinin and recoverin. Our results suggest that neurocalcin may play an important role in a Ca2+ signal pathway of the nervous system.
Animals↗
Full sequence of neurocalcin, a novel calcium-binding protein abundant in central nervous system.
We determined the cDNA sequence for neurocalcin, a novel calcium-binding protein in bovine brain. This clone (pCalN) has 582 nucleotides in the open reading frame including the termination codon TGA, 11 nucleotides of the 5' leader and 1251 nucleotides of the 3' noncoding region. The deduced amino acid sequence revealed that neurocalcin is composed of 193 amino acids, has a molecular mass of 22,284 daltons, and contains three putative calcium-binding sites (EF-hand motifs). By Northern blot analysis, 3.8kbp mRNA was detected in brain. The deduced amino acid sequence had a strong homology to visinin (46.5%) and recoverin (51.6%) in retina, suggesting that neurocalcin may play a visinin- or recoverin-like role in brain.
Amino Acid Sequence↗
Suppression of the Arthus reaction by Y-24180, a potent and specific antagonist of platelet-activating factor.
A novel and potent antagonist of platelet-activating factor (PAF), Y-24180 (4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4] diazepine) was investigated for the effects on the skin reactions induced by chemical mediators and the Arthus reactions. In the rat dorsal skin, Y-24180 (0.1-10 mg/kg, p.o.) inhibited increase in vascular permeability by the intradermal PAF injection in a dose dependent manner and the inhibitory activity was 60 times more potent than that of WEB 2086. While even at doses as large as 10 mg/kg, p.o., it had no effect on vascular permeability in the rat skin induced by histamine, serotonin, bradykinin and leukotriene D4. On a reversed passive Arthus reaction in rat dorsal skin, Y-24180 (0.1-1 mg/kg, p.o.) markedly inhibited vascular permeability in a dose dependent manner and the inhibitory activity was 15 times more potent than that of WEB 2086. Y-24180 also inhibited the Arthus dermal reaction in rabbits (0.03-0.3 mg/kg, p.o.) and guinea pigs (0.1-1 mg/kg, p.o.). In addition, Y-24180 (0.1-10 mg/kg, p.o.) significantly reduced the exudate volume and the number of infiltrated inflammatory cells in the reversed passive Arthus pleural reaction in rats. Furthermore, in rat passive Arthus pancreatitis, Y-24180 (0.3-10 mg/kg, p.o.) significantly inhibited the dye extravasation from the pancreas. These results provide strong evidence that endogenous PAF plays an important role as a mediator in the type III allergic inflammation.
Animals↗
Enhancement of host defense by Y-25510, (+-)-3-[4-(2-dimethylamino-1-methylethoxy)phenyl]-1H-pyrazolo[3,4 -b] pyridine-1-acetic acid, a novel synthetic compound. A comparison with recombinant human granulocyte colony-stimulating factor in 5-fluorouracil-treated mice.
The effect of a novel synthetic compound, Y-25510, (+-)-3-[4-(2-dimethylamino-1-methylethoxy)phenyl]-1H-pyrazolo[3,4 -b] pyridine-1-acetic acid, on recovery from long-lasting leukopenia induced by 5-fluorouracil was compared with that of recombinant human granulocyte colony-stimulating factor (rhG-CSF). When mice were administered i.p. with 5-FU (200 mg/kg) on days 0 and 7, intravenous administration of Y-25510 (100 and 1000 micrograms/kg) prevented the decrease in the peripheral leukocyte and neutrophil number and accelerated the recovery from leukopenia. Subcutaneous administration of rhG-CSF (50 micrograms/kg) did not prevent leukopenia but accelerated the recovery from leukopenia. In particular, peripheral neutrophil number increased over a normal level. The administration of Y-25510 (10, 100 and 1000 micrograms/kg) restored the decrease in the number of bone marrow cells, spleen cells, lymphocytes, neutrophils and monocytes. The administration of rhG-CSF (50 micrograms/kg) restored the decrease in the number of bone marrow cells, spleen cells, and neutrophils but not that of lymphocytes and monocytes. In fractions of bone marrow cells on day 21, the administration of Y-25510 (1000 micrograms/kg) showed a tendency of restoring the decrease in neutrophil number. In conclusion, the administration of Y-25510 prevented leukopenia and accelerated the recovery from leukopenia in the 5-FU-treated mice. It is suggested that the mechanism of the restorative action of Y-25510 is different from that of rhG-CSF. In a number of immature bone marrow cells Y-25510 has a potent stimulatory effect on the recovery from the decrease in number of hematopoietic cells, keeping a balance in number of each blood cell.
Adjuvants, Immunologic↗
Application of the tryptophanase promoter to high expression of the tryptophan synthase gene in Escherichia coli.
The application of an inducible regulation system using the tryptophanase operon promoter (TPase promoter; Ptna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of Ptna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, L-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-Ptna TS was increased 500-fold compared to that of the cultured host strain. About 1 mol L-tryptophan/l reaction mixture was formed from indole and L-serine at 37 degrees C for 3.5 h.
Carbon↗