A new method for the assay of exposed platelet fibrinogen receptor using a chemiluminescent label.

Assay of the platelet fibrinogen-binding receptor glycoprotein (GP) IIb/IIIa is widely performed using 125I-labeled fibrinogen (125I-fibrinogen). We successfully devised a receptor binding assay system with high selectivity and sensitivity using a stable chemiluminescent acridinium derivative-I-labeled fibrinogen (acridinium-fibrinogen). Human fibrinogen is saline was labeled with equimolar acridinium dissolved in dimethylformamide, and allowed to react with gel-filtered human platelets in the presence of ADP. Acridinium-fibrinogen binding to GPIIb/IIIa was assayed by measuring chemiluminescence emitted on addition of 0.1 N NaOH containing 0.06% H2O2 in a luminometer. Non-specific binding was measured in the presence of 10 mM EDTA. Acridinium-fibrinogen binding to human platelets was rapid and reversible, specific and saturable, and dependent on ADP concentrations. Scatchard plot analysis revealed one class of binding sites with Kd of 326 nM and Bmax of 7.8 pmol/10(8) platelets. These values were comparable to the data obtained by using 125I-fibrinogen. Unlabeled fibrinogen, RGDS, and HHLGGAKQAGDV (fibrinogen gamma-chain 400-411) displaced acridinium-fibrinogen from its binding site with Ki values of 322 nM, 9.2 microM and 31.3 microM, respectively. Thus, this binding assay system may be useful in measuring the binding between platelet GPIIb/IIIa and fibrinogen without using a radioisotope.

Ecabet sodium, a novel locally-acting anti-ulcer agent, protects the integrity of the gastric mucosal gel layer from pepsin-induced disruption in the rat.

BACKGROUND: Ecabet sodium, a novel non-systemic anti-ulcer agent, possesses high affinity to gastric adherent mucus, which plays an important role in the protection of the gastric epithelium against acid and pepsin. AIM: To assess the effect of ecabet on pepsin-induced degradation of the structure of the mucus gel layer. METHODS: Everted sacs of rat stomach were incubated in HCl solution containing pepsin with or without ecabet. Pepsin-induced release of the cleaved peptides and hexosamine from the sacs was determined. Changes in the molecular size of glycoproteins in the adherent mucus (using gel filtration methods) and in the morphology of the epithelium (using both light and scanning electron microscopy) were also examined. RESULTS: Ecabet reduced the pepsin-induced release of peptides and hexosamine, depending on its content in the adherent mucus. Pepsin treatment partially lowered the molecular weight of native glycoproteins in the adherent mucus, caused exfoliation of the epithelial cells, and degraded the network-like ultrastructure of the mucus layer, giving it a lumpy, globular appearance. Ecabet prevented both the pepsin-induced molecular size shift in mucus glycoproteins, and morphological alteration of the epithelium, including ultrastructural derangement of the mucus gel layer. CONCLUSION: Ecabet protects the polymeric structure of mucus glycoproteins from proteolytic degradation by pepsin, and thus maintains integrity of the gastric mucus gel layer.

A radioimmunoassay for TA-0910, a new metabolically stable thyrotrophin-releasing hormone analogue.

TA-0910 [1-methyl-(S)-4,5-dihydroorotyl-L-histidyl-L-prolineamide] is a metabolically stable analogue of thyrotrophin-releasing hormone (TRH) and is under clinical investigation as a central nervous system function modulator. A method for determination of its plasma concentrations by radioimmunoassay (RIA) was established. TA-0910 was conjugated to bovine serum albumin and keyhole limpet haemocyanin (KLH) with bis-diazotized benzidine and 1,5-difluoro-2,4-dinitrobenzene as bridging agents. Anti-TA-0910 antisera were prepared by immunizing rabbits with the TA-0910 conjugates and Freund's complete adjuvant. The radiolabelled TA-0910 for RIA was prepared by introducing 125I into the histidine imidazole ring of TA-0910 by the Na125I/chloramine-T method, and purified by reversed phase high-performance liquid chromatography to give a specific radioactivity of 81.4 TBq mmole-1. As the result of testing the cross-reactivity of the antisera with assumed TA-0910 metabolites and TRH, a TA-0910-selective antiserum was obtained from a rabbit immunized with TA-0910-dinitrophenyl-KLH. RIA using this antiserum and the radiolabelled TA-0910 afforded a determination range of 10 pg approximately 5 ng ml-1 plasma. By using this RIA, the time courses of plasma concentrations of unchanged TA-0910 after oral and intravenous administration of TA-0910 were obtained in rats.

The male-specific factor Sry harbors an oncogenic function.

Sgf29, a component of the SPT-ADA-GCN5 acetyltransferase (SAGA) complex, binds H3K4me2/3 marks and leads to histone H3 acetylation. Previously, we found that downregulation of Sgf29 suppresses c-Myc-mediated malignant transformation. Nonetheless, the upstream regulator of the Sgf29 gene is not yet known. Here, we report that Sry (sex-determining region Y), an HMG (high-mobility group) domain containing transcription factor, directly upregulates Sgf29 gene expression. Sry expression was deregulated in two out of the four tested male rodent hepatocellular carcinoma (rHCC) cell lines. Luciferase reporter and chromatin immunoprecipitation assays indicated that Sry could bind HMG-boxes in the proximal promoter region of the Sgf29 gene. Knockdown of Sry robustly lowered anchorage-independent growth, invasiveness and tumorigenicity of rHCC cells, whereas ectopic expression of Sry conferred more malignant properties. Thus, these data show that Sry is involved in male-specific malignant conversion of rHCCs via Sgf29 upregulation.

Studies of metabolic pathways of trimebutine by simultaneous administration of trimebutine and its deuterium-labeled metabolite.

Trimebutine maleate (I), (+-)-2-dimethylamino-2-phenylbutyl 3,4,5-trimethoxybenzoate hydrogen maleate, and a deuterium-labeled sample of its hydrolyzed metabolite, 2-dimethylamino-2-phenylbutanol-d3 (II-d3), were simultaneously administered to experimental animals at an oral dose of 10 or 50 mumol/kg, and distribution ratios of the two alternative initial metabolic steps, i.e., ester hydrolysis and N-demethylation, were estimated by determining the composition of the urinary alcohol-moiety metabolites, II, and its mono- and di-demethylated metabolites, III and IV, by GC/MS. In dogs, the order of quantities of the metabolites from II-d3 was II much greater than III much greater than IV, showing predominance of conjugation over N-demethylation. However, this order was reversed when the amounts of the metabolites from I were compared, indicating that I was preferentially metabolized by N-demethylation followed by ester hydrolysis and conjugation in this order. In rats, a considerable proportion of I was presumed to be metabolized by ester hydrolysis before N-demethylation. In in vitro experiments employing the liver microsomes and homogenates of liver and small intestine from rats and dogs, it was found that both ester-hydrolizing and N-demethylating activities were higher in rats than in dogs, and the conjugating activity was higher in dogs than in rats. It was also found that I, having a high lipophilicity, was more susceptible to N-demethylation than less lipophilic II. These results from the in vitro experiments could account for the species differences in the distribution ratio of the metabolic pathways of I in vivo.

Imidapril, an angiotensin-converting enzyme inhibitor, inhibits thrombosis via reduction in aortic plasminogen activator inhibitor type-1 levels in spontaneously hypertensive rats.

It is known that angiotensin II (Ang II) exerts an antifibrinolytic effect by stimulating synthesis of plasminogen activator inhibitor type-1 (PAI-1), a specific inhibitor of tissue plasminogen activator (t-PA). The aim of this study was to compare the antithrombotic potency of imidapril, an angiotensin-converting enzyme (ACE) inhibitor, and candesartan, an angiotensin II type 1 (AT1) receptor antagonist, in a model of arterial thrombosis in spontaneously hypertensive rats (SHRs). Oral treatment with 5 mg/kg imidapril 1 h before induction of thrombosis resulted in a significant reduction in thrombus weight, whereas candesartan did not affect thrombus weight under the same treatment conditions. Candesartan lowered blood pressure to the same degree as in the imidapril-treated rats. Imidapril not only reduce the serum and aortic ACE activities, but also reduced aortic PAI-1 protein levels, while candesartan had no effect on theses. These results suggest that imidapril, but not the AT1 receptor antagonist, candesartan, enhances fibrinolysis via a reduction of aortic PAI-1 levels by inhibiting ACE and prevents thrombus formation in SHRs.

Angiotensin-converting enzyme inhibitor prevents plasminogen activator inhibitor-1 expression in a rat model with cardiovascular remodeling induced by chronic inhibition of nitric oxide synthesis.

Plasminogen activator inhibitor-1 (PAI-1) may participate in the development of cardiovascular remodeling by inhibiting extracellular matrix turnover and fibrinolysis. However, little is known about physiological regulators of PAI-1 in vivo. Angiotensin II has been shown to stimulate PAI-1 in vitro. We previously reported that long-term inhibition of nitric oxide (NO) synthesis with Nomega-nitro-L-arginine methyl ester (L-NAME) causes cardiovascular remodeling (vascular medial thickening and fibrosis) associated with increased tissue angiotensin-converting enzyme (ACE) activity. In the present study, we examined whether treatment with an ACE inhibitor modulates the cardiovascular PAI-1 expression in this model in vivo. Wistar-Kyoto rats were treated with either no drugs, L-NAME (100 mg/kg x day), or L-NAME plus the ACE inhibitor imidapril (20 mg/kg day). Marked increases in PAI-1 mRNA and protein levels in the aorta and left ventricle were observed after the first and fourth weeks of PAI-1 treatment. PAI-1 immunoreactivity was increased in the endothelium and the media of the aorta and coronary arteries after treatment of L-NAME. This increase in PAI-1 levels was associated with an increase in ACE activity of the aorta and left ventricle. ACE inhibition with imidapril significantly prevented both the increases in PAI-1 levels and the development of cardiovascular remodeling. These findings suggest that the local renin-angiotensin system regulates PAI-1 expression, and that the increased PAI-1 levels may contribute to the cardiovascular remodeling in this model.

Dynamics of antigen-specific helper T cells at the initiation of airway eosinophilic inflammation.

Bronchial asthma is characterized by chronic eosinophilic inflammation of the bronchial mucosa in which Th2 cells play crucial roles. Ovalbumin-reactive Th2 clones were labeled with a fluorescent-probe then infused into unprimed mice to elucidate the dynamics of antigen-specific T cells involved in allergic inflammation. Infiltration of not only labeled antigen-specific T cells, but also unlabeled nonspecific CD4(+) T cells into the bronchial mucosa following inhaled antigen challenge was detectable under confocal microscopy and flow cytometry. Accordingly, labeled T cells in the spleen were decreased, whereas those in hilar lymph nodes were increased upon antigen challenge. Approximately 45% of antigen-specific T cells that migrated into the lungs bore CD25, while another early activation marker, CD69, was expressed on 80% of the migrated T cells. Accordingly, antigen challenge to the mice induced in situ proliferation of antigen-specific T cells as well as bronchial epithelial cells in the lungs. Expression of vascular cell adhesion molecule (VCAM)-1, but not intercellular adhesion molecule (ICAM)-1, on the vascular endothelium in the lungs was enhanced following antigen challenge. Nevertheless, treatment with anti-VCAM-1 antibody, and also anti-ICAM-1 antibody strongly suppressed the accumulation of T cells, suggesting that both VCAM-1 and ICAM-1 are essential for antigen-stimulated T cell mobilization into peripheral tissues. Our current study visualized the kinetics and the mechanism of antigen-specific T cell migration in response to local challenge with a protein antigen.