Simultaneous assay of activated platelet count and platelet-activating capacity by P-selectin detection using K2-EDTA-treated whole blood for antiplatelet agents.

INTRODUCTION: It is well recognized that examinations of activated platelets (aPLTs) and platelet-activating capacity are very important to observe and prevent embolic diseases (events) such as ischemic stroke and myocardial infarction. Previously, we reported an appropriate measurement technique of aPLT for clinical assay. In this paper, we investigated stable conditions for measurement of activating capacity of platelets. METHODS: Blood samples were taken from healthy volunteers using anticoagulants of 2K-EDTA, sodium citrate and heparin, and platelets were stimulated with adenosine diphosphate (ADP) or collagen. We demonstrated platelet-activating capacity by detection of scattering light, absorbance, microscopic observation, and P-selectin (CD62P) expression. We also performed basic experiments in seven healthy volunteers to test the clinical application of these assays with monitoring aspirin therapy. RESULTS: We judged that samples of whole blood with 2K-EDTA were suitable for CD62P expression assay as functional assessments of platelet activity, because platelets treated with anticoagulants such as sodium citrate and heparin were extremely damaged after stimulation, and it was difficult to measure the CD62P expression by flow cytometry. For optimal results, samples should be tested within 1 h after the drawing of blood and stimulated with ADP or collagen for 10 min. The CD62P-positive platelet value of blood from volunteers who had taken aspirin was decreased, and platelet activation was inhibited as well. CONCLUSION: The simultaneous assay of aPLT and platelet-activating capacity by CD62P detection using whole blood treated with the K2-EDTA anticoagulant was useful for the monitoring of antiplatelet drugs.

Isolation and expression of an ecdysteroid-inducible neutral endopeptidase 24.11-like gene in wing discs of Bombyx mori.

In the process of comparison of two cDNA libraries (W0, W2), we isolated a clone from the wing discs of Bombyx mori encoding a putative neutral endopeptidase 24.11-like gene. The predicted open reading frame encoded 772 amino acid residues, having about 53% identity with Drosophila GH07643, 36% with rat NEP, and 34% with rat ECE. This is the first NEP gene isolated in invertebrate. A 3.6-kb transcript was found to accumulate in the wing disc according to the increase of ecdysteroid titer during metamorphosis. Accumulation of the transcript was induced in wing discs with 20-hydroxyecdysone about 20h after incubation, which was inhibited by cycloheximide. This gene is ecdysone-inducible, appears to encode a functional protein, and may function during wing metamorphosis.

Involvement of spinal substance P and excitatory amino acids in inflammatory hyperalgesia in rats.

To reveal possible involvement of NK-1 substance P receptors and N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in the production of inflammatory hyperalgesia, we examined the effects of intrathecal injections of antagonists at those receptors on the nociceptive threshold of inflammatory hyperalgesic rats in the paw-pressure test. Intrathecal injections of the NK-1 antagonist CP-96,345 (0.3-3 nmol/rat), the NMDA antagonist D-2-amino-5-phosphonovaleric acid (D-APV, 1-10 nmol/rat), and the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 1-10 nmol/rat) dose-dependently suppressed adjuvant- and carrageenin-induced hyperalgesia, without effect on the nociceptive threshold of non-inflamed paws. Furthermore, to estimate whether inflammatory hyperalgesia is accompanied with an alteration of the responsiveness to substance P and excitatory amino acids, we examined the effects of injections of complete Freund's adjuvant (intradermal) and carrageenin (subcutaneous) on the aversive responses to intrathecal substance P and excitatory amino acid agonists. Both injections significantly potentiated the aversive behaviors elicited by intrathecal injections of excitatory amino acid agonists, NMDA (1 nmol/rat), a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA, 1 nmol/rat) and kainate (1 nmol/rat), but not those by substance P. The present results suggest that the enhancement of synaptic transmission mediated by substance P and excitatory amino acids in the spinal dorsal horn is at least partly involved in the production of inflammatory hyperalgesia, and that such a hyperalgesia is accompanied with the enhanced responsiveness to excitatory amino acids through NMDA and non-NMDA receptors, but not with changes in responsiveness to substance P.

Effect of repeated cold stress on capsaicin-evoked release of glutamate from rat spinal dorsal horn slices.

We investigated the effect of repeated cold stress (RCS) on the capsaicin-evoked release of glutamate from the primary afferent fibers of the rat, and compared this with the effect of inoculation of complete Freund's adjuvant (adjuvant inoculation). The release of glutamate was measured using a fluorometric on-line continuous monitoring system in which the immobilized glutamate dehydrogenase column was connected to an in vitro superfusion system. In the presence of 0.3 microM tetrodotoxin, the application of 1 microM capsaicin to spinal dorsal horn slices evoked glutamate release (18.6 +/- 1.2 pmol mg(-1) protein, n = 11). In rats subjected to RCS (RCS rats), the release of glutamate evoked by 1 microM capsaicin was markedly increased to 272% (n = 6, P < 0.05) of the value for the control group, although the basal release was not significantly altered (n = 6, P > 0.05). Adjuvant inoculation produced a significant increase in the basal and capsaicin (1 microM) evoked release of glutamate to 141 and 344% (n = 6, P < 0.05) of the value for the control group, respectively. The present results suggest that the facilitated release of glutamate from capsaicin-sensitive primary afferent terminals in the spinal dorsal horn is, at least in part, involved in the hyperalgesia of RCS rats as well as the complete Freund's adjuvant-induced hyperalgesia.

Protective effects of antithrombin III supplementation on warm ischemia and reperfusion injury in rat liver.

The effect of antithrombin III (AT III) supplementation on energy status, microcirculation, cytoprotection, and prostacyclin (PGI2) production during and after a period of warm ischemia of the rat liver was investigated. AT III supplementation (250 units/kg) stimulate prostaglandin I2 (PGI2) production from 1 hour after administration, with maximal production observed at 3 hours. Ischemia was induced by occluding the hepatoduodenal ligament for 30 minutes, and experiments were continued for 60 minutes after reperfusion. The rats received AT III (250 units/kg IC) 30 minutes before induction of liver ischemia (AT III group). In the AT III group, recovery of the beta-ATP/inorganic phosphate ratio measured by 31P nuclear magnetic resonance showed significant improvement (p < 0.01), and the recovery of tissue blood flow markedly improved (p < 0.01) compared to the saline-treated group (control group). Leakages of aspartame aminotransferase, alanine aminotransferase, and lactate dehydrogenase were mitigated in the AT III group (p < 0. 05). Ultrastructural alterations of sinusoidal endothelial cells were markedly reduced in the AT III group. The PGI2 level at the end of reperfusion was significantly elevated (p < 0.01) in the AT III group compared to the control group. The results of this study indicated that pretreatment with AT III significantly improved the energy status and microcirculation, as well as histologic damage, after liver ischemia and reperfusion. One of the fundamental effects of AT III might be mediated through the production of prostacyclin.

Parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor and its messenger ribonucleic acid in rat aortic vascular smooth muscle cells and UMR osteoblast-like cells: cell-specific regulation by angiotensin-II and PTHrP.

PTH-related protein (PTHrP) is produced in vascular smooth muscle, where it is believed to act as a local vasorelaxant by activating either the classical PTH or a unique PTHrP receptor. We used a newly cloned complementary DNA encoding the rat PTH/PTHrP receptor to study the expression of its messenger RNA (mRNA) in primary aortic vascular smooth muscle cells (VSMC) and in UMR-106 osteoblast-like cells under basal conditions and in response to treatment with agonists. Both cell types expressed a 2.4-kilobase PTH/PTHrP receptor mRNA transcript and exhibited hormone-induced desensitization of PTHrP-(1-34)NH2-stimulated cAMP. In VSMC, angiotensin-II, which induces PTHrP expression, also rapidly (30 min) desensitized the cAMP response and down-regulated (75-90%) receptor mRNA within 1 h. Treatment of cells with phorbol 12-myristate 13-acetate (0.1 microM) mimicked these effects, whereas neither PTHrP-(1-34)NH2, forskolin, nor (Bu)2cAMP altered receptor mRNA expression. By contrast, in UMR-106 cells, PTHrP-(1-34)NH2 induced time- and dose-dependent decreases in receptor mRNA that were preceded by pronounced desensitization (cAMP and ligand binding) of cell surface receptors. These effects were mimicked by (Bu)2cAMP and forskolin, but not by phorbol 12-myristate 13-acetate, suggesting that both receptor mRNA down-regulation and receptor desensitization in UMR cells were mediated through a protein kinase-A pathway. We suggest that VSMC and UMR cells express a common receptor, which is subject to cell-specific regulation. Such diversity in the PTH/PTHrP receptor regulatory mechanisms provides a means for restricting the length and duration of the cellular response to hormone in a cell/tissue-specific manner.

Molecular cloning of a novel receptor tyrosine kinase gene, STK, derived from enriched hematopoietic stem cells.

To identify the novel receptor tyrosine kinases (RTKs) critical to the proliferation of hematopoietic stem cells, we performed polymerase chain reaction-based cloning from highly purified murine hematopoietic stem cells. Lineage marker-negative, c-KIT-positive, and Ly6A/E- or Sca-1-positive (Lin-c-KIT+Sca-1+) cells were sorted by a fluorescence-activated cell sorter. Two sets of degenerate oligonucleotide primers were directed to the conserved sequences of the catalytic domain, and were used to amplify cDNAs that encode protein tyrosine kinases (PTKs). One hundred cDNA clones were sequenced and 8 RTKs were identified, as well as 12 non-RTKs and 2 serine/threonine kinases. Sixteen cDNAs were identical to the known kinase genes (PKC beta, JAK-1, JAK-2, TYK-2, HCK, FGR, FYN, BLK, c-FES, FER, c-ABL, c-KIT, FLK-1, FLK-2, IGF1R, and ECK). Six novel cDNA sequences (stk series) were identified. However, three of them turned out to be BPK, RYK, and TEK. The remaining three showed high homology to S6 kinase II, JAK-2, and v-SEA/c-MET, respectively. Characterization of full-length cDNA sequence of the v-SEA/cMET-related gene showed that this was a novel RTK gene and we named this gene STK (stem cell-derived tyrosine kinase). We identified two distinct forms of STK cDNA; the short one encoded a putative truncated protein that lacked most of the extracellular domain. STK was expressed at various stages of hematopoietic cells, including stem cells, but we could not detect any apparent expression in other adult tissues. The expression of the truncated form of mRNA was more predominant than that of the complete form. STK was assigned by fluorescent in situ hybridization to the R-positive F1 band of chromosome 9, the same region to which hepatic growth factor-like protein has been assigned. Characterization of these PTKs, including STK, will be helpful to elucidate the molecular mechanism of the growth regulation of hematopoietic stem cells.

[A case of malignant fibrous histiocytoma of the cecum].

A case of primary malignant fibrous histiocytoma (MFH) of the cecum was reported. Patient was a 52-year-old female, and complained of right lower abdominal pain. The barium enema and abdominal computed tomography demonstrated a localized mass involving the entire circumference of the cecum. Right hemicolectomy was performed and the resected specimen revealed a tumor of 8 X 6 X 5 cm in size extending to the entire circumference of the cecum. The histopathological examination revealed a storiform pattern, which was diagnostic of MHF. The tumor proliferated chiefly in the subserous tissue and partially infiltrated the tunica muscularis propria. The postoperative course was uneventful and she showed no sign of recurrence at 15 months after operation. This is the ninth reported case of primary MFH in digestive organs.

Antiulcerogenic compounds isolated from Chinese cinnamon.

Two active compounds that prevent serotonin-induced ulcerogenesis in rats were isolated from Chinese cinnamon (the stem bark of Cinnamomum cassia) and identified as 3-(2-hydroxyphenyl)-propanoic acid and its O-glucoside. The former compound, administered orally or parenterally to rats at a remarkably low dose (40 micrograms/kg body weight), also inhibited gastric ulcers induced by the other ulcerogens such as phenylbutazone, ethanol, and water immersion stress, although it failed to prevent indomethacin-induced ulcers. Pharmacological studies have shown that 3-(2-hydroxyphenyl)-propanoic acid hardly inhibited the secretion of gastric acid, but promoted the gastric blood flow. These results suggest that the antiulcerogenic effect of this compound is probably attributable to the potentiation of defensive factors through the improvement of the circulatory disorder and gastric cytoprotection.

Modulation of hepatotoxicity by macrophages in the liver.

In an attempt to elucidate the role of hepatic macrophages in liver injury, we investigated galactosamine-treated rats (500 mg per kg body weight). The rats received an i.v. injection of latex particles (2 x 10(9) particles per animal) prior to (latex-galactosamine) or 12 to 16 hr subsequent to the galactosamine treatment (galactosamine-latex). Effect of superoxide dismutase on hepatic injury induced by galactosamine or galactosamine-latex treatment was also examined. Oxygen-derived free radical-generating capacity of isolated hepatic macrophages was measured as chemiluminescence with the stimulation of phorbol myristate acetate or latex particles. As compared with normal rats, chemiluminescence of hepatic macrophages from galactosamine-treated rats was 5- to 10-fold enhanced 12 hr following galactosamine treatment and remained elevated for 48 hr. Chemiluminescence of the latex particle-pretreated macrophages in the liver was markedly suppressed even following the galactosamine treatment (p less than 0.01). Compared to galactosamine-treated rats, both lipid peroxide level in the liver tissue and AST and ALT concentration in serum were significantly decreased in the latex-galactosamine-treated rats (p less than 0.01) and increased in the galactosamine-latex-treated rats (p less than 0.01). Furthermore, superoxide dismutase supplementation protected against liver injury induced by the galactosamine-latex treatment. From these results, pretreatment with latex particles suppressed the free radical-generating capacity of hepatic macrophages and protected against hepatic injury induced by galactosamine. In contrast, injection of latex particles after galactosamine treatment aggravated hepatic injury, which was prevented by superoxide dismutase. These data suggest that liver injury induced by galactosamine is modulated by oxygen-derived free radicals from hepatic macrophages.

Comparative efficacy of various vitamin D metabolites in the treatment of various types of hypoparathyroidism.

Fourteen patients with pseudohypoparathyroidism, 17 with idiopathic hypoparathyroidism, and 12 with postoperative hypoparathyroidism were treated with vitamin D2, dihydrotachysterol, 1 alpha-hydroxyvitamin D3)1 alpha-OHD3), and 1,25-dihydroxyvitamin D3 for 6-18 months. The optimal maintenance dose or minimum daily dose of 1,25-dihydroxyvitamin D3 to maintain serum calcium at approximately 8.5 mg/100 ml and control all the clinical symptoms was 1.3 +/- 0.16 micrograms/day (mean +/- SE) in pseudohypoparathyroidism, 1.5 +/- 0.18 micrograms/day in idiopathic hypoparathyroidism, and 1.9 +/- 0.50 micrograms/day in postoperative hypoparathyroidism. There was no significant difference in the optimal maintenance dose among the 3 groups. The optimal maintenance dose of 1 alpha-OHD3, however, was 2.0 +/- 0.12 micrograms/day in pseudohypoparathyroidism, significantly lower than that in idiopathic hypoparathyroidism (3.5 +/-0.29 micrograms/day; P less than 0.001) and in postoperative hypoparathyroidism (4.89 +/- 0.54 micrograms/day; P less than 0.001). Significantly lower doses were required in the treatment of idiopathic hypoparathyroidism than in postoperative hypoparathyroidism (P less than 0.05). No significant difference was found in the optimal maintenance dose of dihydrotachysterol and vitamin D2 among the 3 groups. The average pretreatment serum calcium levels and clinical manifestations were indistinguishable among the 3 groups of patients. This suggests that such a difference in the optimal maintenance dose of 1 alpha-OHD3 is ascribed not to the difference in the severity of hypoparathyroidism, but most probably to differences in the pathophysiological processes in pseudohypoparathyroidism and idiopathic or postoperative hypoparathyroidism. The excess parathyroid hormone levels in blood of patients with pseudohypoparathyroidism (and not in other types of hypoparathyroidism) may explain such a difference.