Pharmacological effects of oral E6123, a novel PAF antagonist, on biological changes induced by PAF inhalation in guinea pigs.

The effects of a newly synthesized PAF antagonist E6123, (S)-(+)-6-(2-chlorophenyl)-3-cyclopropanecarbonyl-8,11-dimethyl-2, 3,4,5- tetrahydro-8H-pyrido[4',3':4,5]thieno[3,2-f][1,2,4]triazolo [4,3-a][1,4]diazepine, on in vivo inhaled PAF-induced pulmonary changes were investigated. E6123 inhibited PAF inhalation-induced bronchoconstriction in guinea pigs with an ED50 value (p.o.) of 1.3 micrograms/kg which was lower than those of other PAF-antagonists such as WEB2347 (ED50 = 26 micrograms/kg) and Y-24180 (ED50 = 12 micrograms/kg). E6123 significantly inhibited PAF inhalation-induced eosinophil infiltration into the bronchiole and trachea, and bronchial hyperreactivity in guinea pigs after oral administration at 1 and 10 micrograms/kg, respectively. E6123 inhibited the PAF-induced increase in intracellular free calcium ion concentration ([Ca2+]i) in guinea pig eosinophils with an IC50 value of 14 nM. The present results suggest that E6123 may be beneficial for the treatment of asthma, in which PAF is assumed to be involved.     

Involvement of leukotriene C4 in PAF-induced death in mice

The role of leukotrienes (LTs) in the pathogenesis of platelet-activating factor (PAF)-induced death in mice was reinvestigated, since previously reported results are in conflict. A novel 5-lipoxygenase inhibitor, E6080, and a leukotriene antagonist, LY17883, protected mice from PAF-induced death in a dose-dependent manner, while the well-known 5-lipoxygenase inhibitor, AA861, was less effective than E6080. After the intravenous injection of PAF in mice, immunoreactive leukotriene C4 (i-LTC4), which was co-eluted with authentic LTC4 in HPLC, was significantly increased in bronchoalveolar lavage fluid (BALF). Oral administration of E6080 suppressed the increase in i-LTCM4. The results suggest that LTs may play an important role in PAF-induced lethality in mice.     

Movement of vesicles in cytoplasmic streaming in plasmodium

The moving velocities of vesicles in the cytoplasmic streaming of a slime mold were measured, in which all of the vesicles passing through a designated window were counted. Vesicles in the streaming are distributed in their moving velocities and the distribution itself varies with time. The mean velocity of vesicles and its standard deviation were found to exhibit a linear relationship, suggesting a possibility that vesicles in the cytoplasm would also be involved in force generation.     

Triacylglycerol lipase mediated release of arachidonic acid for prostaglandin synthesis in rabbit kidney medulla microsomes

The effect of triarachidonin on the synthesis of prostaglandins in rabbit kidney medulla microsomes was examined. Medulla microsomes were incubated with triarachidonin in 0.1 M--Tris/HCl buffer (pH 7.0) containing reduced glutathione and hydroquinone and the formed prostaglandin E2, prostaglandin F2 alpha and prostaglandin D2 were measured by high-pressure liquid chromatography using 9-anthryldiazomethane for derivatization. The addition of triarachidonin (1-10 microM) stimulated prostaglandin formation in a dose-dependent manner. Under our incubation conditions rabbit kidney medulla was found to produce prostaglandin E2 mainly. When arachidonic acid, instead of triarachidonin, was added to the incubation mixture of microsomes, the identical profile of prostaglandin products was obtained. When the pH of the reaction mixture was changed from 7.0 to 8.0, the rate of triarachidonin-induced prostaglandin E2 formation was approximately 60% of that observed at pH 7.0. Studies utilizing Ca2+ and EGTA revealed that triacylglycerol lipase of kidney medulla is independent of Ca2+. The addition of epinephrine made the stimulatory effect of triarachidonin on prostaglandin E2 formation more pronounced. These results suggest that epinephrine-activated triacylglycerol lipase is present in the renomedullary microsomes, and this enzyme activity is a potential mediator of release of arachidonic acid for prostaglandin synthesis in the kidney medulla.     

Evaluating the occurrence of cryptic invasions of a rocky shore barnacle,Semibalanus cariosus,between the north-eastern Pacific and Japan

Many coastal barnacles are introduced to non-native regions. However, data are lacking on cryptic invasion, which is defined as an invasion that remains unrecognised because the invader is mistaken for a native or previously introduced species or clade. In this work, cryptic invasions of an intertidal barnacle, Semibalanus cariosus, between Japan and the north-eastern Pacific were evaluated based on population genetic analyses. A significant genetic differentiation was found between the Japanese and north-eastern Pacific populations, suggesting a limited introduction of non-native genotypes between these regions. Haplotype frequencies did not differ significantly between the past (museum samples collected in 1971 from Hokkaido, Japan) and present Japanese populations, implying the rare occurrence of human-mediated migration from the north-eastern Pacific to Japan. Migrate-n analysis revealed a low level of directional gene flow in S. cariosus from the north-eastern Pacific to Japan, possibly by natural stepping-stone dispersal via directional water currents or human-mediated transport.     

A Di-leucine signal in the ubiquitin moiety. Possible involvement in ubiquitination-mediated endocytosis

Some plasma membrane receptors in yeast are known to be internalized and degraded in lysosomes upon ligand-dependent ubiquitination. However, the role of ubiquitination in endocytosis and lysosomal degradation in higher eukaryotes has been controversial. In order to directly assess this question, we investigated the fate of chimeric molecules in which ubiquitin moiety was fused in-frame to the cytoplasmic region of membrane proteins. The chimeric proteins with the wild-type ubiquitin were endocytosed and delivered to lysosomes efficiently. Mutant ubiquitin with lysine-to-arginine substitution could still mediate endocytosis, suggesting that polyubiquitination is not required for the endocytosis. We next searched for the existence of an endocytosis signal(s) in the ubiquitin moiety, and identified a di-leucine signal, Leu(43)-Ile(44). The Leu(43)-Ile(44) sequence mediated endocytosis and lysosomal sorting in a Leu(43)-dependent manner. These results suggest that the di-leucine signal in ubiquitin can be involved in ubiquitination-mediated endocytosis and lysosomal targeting of membrane proteins.     

Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression

In plants, a cis-acting element, DRE/CRT, is involved in ABA-independent gene expression in response to dehydration and low-temperature stress. To understand signal transduction pathways from perception of the dehydration stress signal to gene expression, we characterized a gene family for DRE/CRT-binding proteins DREB2A and DREB2B in Arabidopsis thaliana. Northern analysis showed that both genes are induced by dehydration and high-salt stress. Organ-specific northern analysis with gene-specific probes showed that these genes are strongly induced in roots by high-salt stress and in stems and roots by dehydration stress. The DREB2A gene is located on chromosome 5, and DREB2B on chromosome 3. We screened an Arabidopsis genomic DNA library with cDNA fragments of DREB2A and DREB2B as probes, and isolated DNA fragments that contained 5-flanking regions of these genes. Sequence analysis showed that both genes are interrupted by a single intron at identical positions in their leader sequence. Several conserved sequences were found in the promoter regions of both genes. The -glucuronidase (GUS) reporter gene driven by the DREB2 promoters was induced by dehydration and high-salt stress in transgenic Arabidopsis plants.     

LDL receptor-related protein as a component of the midkine receptor

Midkine (MK) is a heparin-binding growth factor with migration-promoting and survival-promoting activities. To identify signaling receptor(s) of MK, membrane glycoproteins with MK-binding activity were isolated from day 13 mouse embryos by lectin- and MK-affinity chromatography. SDS-PAGE followed by protein sequence analysis revealed the presence of LDL receptor-related protein (LRP) and NCAM in the fraction. The dissociation constant of binding between LRP and MK was 3.5 nM. Receptor-associated protein (RAP), which interfered with the binding, inhibited MK-dependent survival of embryonic neurons. Brushin/megalin, which is also a high molecular weight protein belonging to the LDL receptor family, bound to MK less strongly than LRP. These findings suggest that LRP is a component of the receptor complex for MK.