Pharmacological actions of Y-24180, a new specific antagonist of platelet activating factor (PAF): II. Interactions with PAF and benzodiazepine receptors

The inhibitory effect of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-t hieno [3,2-f][1,2,4]triazolo [4,3-a][1,4]diazepine, on platelet activating factor (PAF)-induced platelet aggregation and the specific binding of 3H-PAF to platelets was compared with other thienodiazepine derivatives, WEB 2086 and etizolam. Y-24180 inhibited PAF-induced rabbit platelet aggregation in vitro (IC50 3.84 nM), but had little effect on adenosine diphosphate- or arachidonic acid-induced aggregation. WEB 2086 and etizolam also showed an inhibitory effect of PAF-induced aggregation (IC50 values are 456 and 6730 nM, respectively). In PAF-induced human platelet aggregation, Y-24180 (IC50 0.84 nM) was more potent than WEB 2086 (IC50 4.21 nM) and etizolam (IC50 998 nM). Y-24180, WEB 2086 and etizolam displaced 3H-PAF binding from the washed-platelets of rabbits with an IC50 value of 3.50, 9.35 and 29.5 nM, respectively. In rabbits, pretreatment with Y-24180 and WEB 2086 antagonized PAF-induced platelet aggregation dose-dependently. The significant inhibitory effect of Y-24180 (1 mg/kg, p.o.) lasted 72 hr after a single dose oral administration. WEB 2086 (10 mg/kg, p.o.) also antagonized the ex vivo response induced by PAF 1 hr after administration, but no significant effect was observed 3 hr after administration. Y-24180 displaced 3H-diazepam binding from the synaptosomal membranes of rat cerebral cortex with a Ki value of 3.68 microM. The affinity of Y-24180 for benzodiazepine(BZP) receptors was lower than those of WEB 2086 and etizolam and was about 1000 times lower than that for PAF receptors in platelets.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments for a chemokine receptor-binding domain of FROUNT,a cytoplasmic regulator of chemotaxis

FROUNT is a cytoplasmic protein that interacts with the membrane-proximal C-terminal regions (Pro-Cs) of the CCR2 and CCR5 chemokine receptors. The interactions between FROUNT and the chemokine receptors play an important role in the migration of inflammatory immune cells. Therefore, FROUNT is a potential drug target for inflammatory diseases. However, the structural basis of the interactions between FROUNT and the chemokine receptors remains to be elucidated. We previously identified the C-terminal region (residues 532–656) of FROUNT as the structural domain responsible for the Pro-C binding, referred to as the chemokine receptor-binding domain (CRBD), and then constructed its mutant, bearing L538E/P612S mutations, with improved NMR spectral quality, referred to as CRBD_LEPS. We now report the main-chain and side-chain ¹H, ¹³C, and ¹⁵N resonance assignments of CRBD_LEPS. The NMR signals of CRBD_LEPS were well dispersed and their intensities were uniform on the ¹H–¹⁵N HSQC spectrum, and thus almost all of the main-chain and side-chain resonances were assigned. This assignment information provides the foundation for NMR studies of the three-dimensional structure of CRBD_LEPS in solution and its interactions with chemokine receptors.     

Continuous ethanol fermentation using flocculent yeast entrapped in Horizontal Parallel Flow bioreactor system

Summary Continuous ethanol fermentations were conducted in single-stage and three-stage Horizontal Parallel Flow (HOPAF) bioreactor systems. Biological entrapment of yeast could be achieved by virtue of its growth and flocculence in reusable porous stainless steel fiber sheets. Twenty-five g·l^–1·h^–1 productivity was obtained in three-stage system. Distributions of ethanol and glucose in reactors were examined.     

Genetic polymorphism of coagulation factor XIII B subunit in the Japanese population: description of three new rare alleles

Polyacrylamide gel isoelectric focusing (PAGIEF) of neuraminidase-treated EDTA plasma samples followed by electroblotting with enzyme immunoassay was performed to further investigate coagulation factor XIII B subunit (FXIII B) polymorphism. In 435 Japanese subjects PAGIEF patterns of FXIII B were classified into five common and three rare allotypes. This suggested that the FXIII B*2 allele existed in the Japanese population in the same manner as in Caucasians. Three new rare allotypes were considered to be controlled by three rare alleles which were designated FXIII B*13, FXIII B*14, and FXIII B*15, respectively. The gene frequencies calculated from 435 Japanese subjects were FXIII B*1 = 0.2977, FXIII B*2 = 0.0184, FXIII B*3 = 0.6805, FXIII B*13 = 0.0011, FXIII B*14 = 0.0011, and FXIII B*15 = 0.0011, respectively.     

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.     

Stereological studies of the parathyroid gland of phosphate-treated golden hamsters subjected to a hypergravity environment.

The ultrastructure of the parathyroid glands of phosphate-treated golden hamsters exposed to a 5-G environment was studied. In the phosphate-treated animals exposed to a hypergravity environment, the Golgi complexes associated with numerous prosecretory granules, and the enlarged intercellular spaces containing floccular or finely particulate material showed a significant increase compared to those of the control, centrifuged, and phosphate-treated groups, and the cisternae of the granular endoplasmic reticulum showed a significant increase compared to those of the control and phosphate-treated groups. In addition, numerous secretory granules were situated close to the plasma membrane of chief cells in the phosphate-treated animals exposed to a hypergravity environment. These findings suggest that the synthesis, and to a greater extent the release of secretory granules may be markedly stimulated, in the parathyroid glands of phosphate-treated animals exposed to a hypergravity environment.     

Pharmacological actions of Y-24180: I. A potent and specific antagonist of platelet-activating factor

The ability 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 to inhibit platelet-activating factor (PAF)-induced reactions was investigated. Y-24180 (0.0003–0.003 mg/kg, i.v.) dose-dependently inhibited PAF-induced bronchoconstriction in guinea pigs, but even at a high dose of 10 mg/kg, i.v., it was either inactive or weakly active against the bronchoconstriction induced by histamine, serotonin, acetylcholine, arachidonic acid, bradykinin, or leukotriene D₄. Oral doses (0.003–0.1 mg/kg) of Y-24180 also prevented hemoconcentration due to PAF in a dosedependent manner and produced a parallel shift of the PAF dose-response curve. Y-24180 (0.0003–0.1 mg/kg, i.v.) and WEB 2086 (0.03–1 mg/kg, i.v.) dose-dependently reversed PAF-induced hypotension in anesthetized rats. In mice, PAF-induced lethality was inhibited by Y-24180 and WEB 2086 with ED₅₀ values of 0.022 and 1.42 mg/kg, p.o., and 0.023 and 0.12 mg/kg, i.v., respectively. This protective effect of Y-24180 given p.o. persisted for at least 6 hr. In actively sensitized mice lethal anaphylactic shock was prevented by oral doses of Y-24180 and WEB 2086 with ED₅₀ values of 0.095 and 0.69 mg/kg, respectively. These results suggested that Y-24180 is an extremely potent and specific PAF antagonist with a good duration of action.     

The LHRH neuronal system in female rats: relation to the medial preoptic nucleus

Previously we have found that small lesions confined to the medial preoptic nucleus (MPN) or the suprachiasmatic nucleus (SCN) blocked the cyclic release of gonadotropins in the female rat, inducing a persistent estrous state. Since the MPN is located just caudal to the organum vasculosum of the lamina terminalis (OVLT) where LHRH cell bodies are most concentrated, we applied an immunocytochemical technique to examine the possibility that the lesions had simply disrupted LHRH neurons or fibers. Using a new anti-LHRH provided by Dr. V. D. Ramirez, we found that the distribution pattern of immunoreactive LHRH cell bodies and fibers was similar to that previously reported, although the staining was more intense and extensive with low background. There was no concentration of LHRH cell bodies and fibers in the MPN or SCN and, in fact, these nuclei generally showed a lower density of stained elements than did surrounding tissue. In persistent estrous animals with lesions confined to the MPN there was no detectable reduction of stained fibers in the median eminence. These results, along with the results of other workers, suggest that persistent estrus following lesions of the MPN or SCN is not due to reduction of LHRH neurons or fibers. Rather, they support the hypothesis that these nuclei are critical for triggering the ovulatory release of LHRH.