Prostaglandin E2 induces contraction of liver myofibroblasts by activating EP3 and FP prostanoid receptors.

BACKGROUND AND PURPOSE: Increased portal pressure in liver injury results from hypercontraction of perivascular non-parenchymal cells including liver myofibroblasts (MFs). Prostaglandin E2 (PGE2) is the major eicosanoid which is released around the venous system during liver injury, but little is known about their contractile effect on MFs. EXPERIMENTAL APPROACH: Contraction of primary rat liver MFs was measured by a collagen gel contraction assay. Expression of E prostanoid (EP) receptor subtypes was assessed by reverse transcription-polymerase chain reaction. Fura-2 fluorescence was used to determine intracellular Ca2+ concentration ([Ca2+](i)). Phosphorylation of protein kinase C (PKC) was detected by Western blot analysis. KEY RESULTS: Liver MFs expressed mRNAs for all four EP receptors. PGE2 induced contraction in a dose- and time-dependent manner, and slightly increased [Ca2+](i) only at high concentrations (10 micromol.L(-1)). An agonist selective for EP(3) receptors, ONO-AE-248, dose-dependently induced MF contraction but did not increase [Ca2+](i). Pretreatment with rottlerin (a specific novel PKC inhibitor) and Ro 31-8425 (a general PKC inhibitor) significantly reduced 1 micromol.L(-1) PGE(2)- or ONO-AE-248-induced contractions. Furthermore, 1 micromol.L(-1) PGE(2) stimulated phosphorylation of PKC isoforms PKCdelta and PKCepsilon. The F prostanoid (FP) receptor antagonist AL8810 abolished the [Ca(2+)](i) elevation and the rapid contraction induced by 10 micromol.L(-1) PGE2. CONCLUSIONS AND IMPLICATIONS: Lower concentrations up to 1 micromol.L(-1) of PGE2 induce liver MF contraction via a [Ca2+](i)-independent PKC-mediated pathway through the EP(3) receptor, while higher concentrations have an additional pathway leading to Ca(2+)-dependent contraction through activating the FP receptor.

Anisotropy and corotation of galactic cosmic rays.

The intensity of Galactic cosmic rays is nearly isotropic because of the influence of magnetic fields in the Milky Way. Here, we present two-dimensional high-precision anisotropy measurement for energies from a few to several hundred teraelectronvolts (TeV), using the large data sample of the Tibet Air Shower Arrays. Besides revealing finer details of the known anisotropies, a new component of Galactic cosmic ray anisotropy in sidereal time is uncovered around the Cygnus region direction. For cosmic-ray energies up to a few hundred TeV, all components of anisotropies fade away, showing a corotation of Galactic cosmic rays with the local Galactic magnetic environment. These results have broad implications for a comprehensive understanding of cosmic rays, supernovae, magnetic fields, and heliospheric and Galactic dynamic environments.

Observation by an air-shower array in Tibet of the multi-TeV cosmic-ray anisotropy due to terrestrial orbital motion around the Sun.

We report on the solar diurnal variation of the galactic cosmic-ray intensity observed by the Tibet III air shower array during the period from 1999 to 2003. In the higher-energy event samples (12 and 6.2 TeV), the variations are fairly consistent with the Compton-Getting anisotropy due to the terrestrial orbital motion around the Sun, while the variation in the lower-energy event sample (4.0 TeV) is inconsistent with this anisotropy. This suggests an additional anisotropy superposed at the multi-TeV energies, e.g., the solar modulation effect. This is the highest-precision measurement of the Compton-Getting anisotropy ever made.

Chalcone isomerase isozymes with different substrate specificities towards 6'-hydroxy- and 6'-deoxychalcones in cultured cells of Glycyrrhiza echinata, a leguminous plant producing 5-deoxyflavonoids.

Three chalcone isomerase isozymes in Glycyrrhiza echinata (Fabaceae) were separated by chromatofocusing and partially purified to examine their substrate specificities. Two isozymes, one of which was elicitor-inducible, acted on both 6'-hydroxychalcone and 6'-deoxychalcone and presumably are involved in the legume-specific 5-deoxyflavonoid pathway, while another acted on only 6'-hydroxychalcone.

Effects of cilostazol on late lumen loss and repeat revascularization after Palmaz-Schatz coronary stent implantation.

BACKGROUND: Cilostazol is an antiplatelet agent that increases the intracellular concentration of cyclic adenosine monophosphate by inhibiting phosphodiesterase III; it has been shown to reduce neointimal hyperplasia in animal balloon injury models. METHODS: One hundred thirty patients who underwent elective stenting (Palmaz-Schatz stent) were randomly assigned to cilostazol treatment 200 mg/d (n = 65) or to ticlopidine treatment 200 mg/d (n = 65). Angiographic follow-up was performed at 6 months, and clinical follow-up was continued up to 1 year. RESULTS: One sudden death and one myocardial infarction resulting from subacute occlusion were observed in the ticlopidine group. Drug adverse effects were observed in 3 patients in the cilostazol group, as opposed to 6 patients in the ticlopidine group. In the intention-to-treat analysis, 56 patients (61 lesions) in the cilostazol group and 58 patients (58 lesions) in the ticlopidine group were assessed with quantitative coronary angiography. Late loss in the cilostazol group was smaller (0.58+/-0.52 mm vs. 1.09+/-0.65 mm, P<.0001) than in the ticlopidine group. The restenosis rate was lower in the cilostazol group than in the ticlopidine group (16% vs. 33%, P = .044). The target vessel revascularization rate at 1 year was 23% in the cilostazol group and 42% in the ticlopidine group (P =.03). CONCLUSIONS: The results of this study suggest that cilostazol may be a safe medication that is effective in preventing restenosis after stent implantation.

New scheme of the biosynthesis of formononetin involving 2,7,4'-trihydroxyisoflavanone but not daidzein as the methyl acceptor.

Glycyrrhiza echinata cell-free extract produced isoformononetin by the 7-O-transmethylation of daidzein from S-adenosyl-L-methionine (SAM). When the yeast microsome expressing 2-hydroxyisoflavanone synthase was mixed with the cell-free extract and incubated with liquiritigenin and SAM, formononetin emerged. Furthermore, the cell-free extract yielded formononetin on incubation with 2,7,4'-trihydroxyisoflavanone and SAM. We propose a novel pathway of formononetin biosynthesis involving 2,7,4'-trihydroxyisoflavanone as the methyl acceptor.

[Gamma-interferon-induced cardiomyopathy during treatment of renal cell carcinoma: a case report].

A 78-year-old woman with renal cell carcinoma and pulmonary metastasis presented with reversible cardiomyopathy induced by gamma(gamma)-interferon. She was treated with gamma-interferon twice a week since November 1996. She presented with severe acute congestive heart failure and gamma-interferon was immediately discontinued in December 1997. Left ventricular fractional shortening was 38% before admission, 12% on admission, and improved to 31% by 40 days after discontinuation of interferon together with administration of diuretics and angiotensin converting enzyme inhibitor. We restarted the same gamma-interferon regimen because it was effective against renal cell carcinoma after 47 days. She has remained well with no significant changes of cardiac function or renal cell carcinoma for almost one year.

Slowly growing cardiac tumor: a case of fibroelastoma.

Echocardiographic follow-up for 16 years in an asymptomatic patient with mitral stenosis showed very slow growth of a mass attached to the mitral valve. The tumor doubling time was estimated to be 3.6 years. Surgical excision of the mass was performed when the patient eventually developed dyspnea on exertion, and histopathological examination revealed papillary fibroelastoma. Echocardiographic follow-up and anti-coagulation may be sufficient treatment for asymptomatic patients with papillary fibroelastoma.

Retrospective analysis of cerebral complications after coronary artery bypass grafting in elderly patients.

The present study retrospectively investigated cerebral complications of coronary artery bypass grafting in 205 consecutive patients aged 70 years or older, who underwent elective cardiopulmonary bypass from 1990 to 1997. Computed tomography of the brain and chest was done before surgery. Ten patients had so-called 'aortic no-touch surgery' and suffered no cerebral complications; the other 195 patients had conventional surgery. Adverse cerebral events occurred in 8.7%, including cerebral infarction (4.1%), diffuse encephalopathy (1.0%), convulsions (1.0%), transient disturbance of consciousness (1.0%), and severe loss of volition (1.5%). Multivariate analysis showed that only the detection of calcification of the ascending aorta was significantly associated with cerebral complications (p = 0.029). Total clamping tended to be superior to partial clamping for prevention of cerebrovascular accidents. The mortality rate was 7.3%. In-hospital death was related to age (p = 0.0062), cerebral complications (p = 0.0032), and a low left-ventricular function (p = 0.018). Therefore, chest computed tomography to assess the ascending aorta should be performed preoperatively. Modified techniques like aortic no-touch surgery or other therapies combined with coronary intervention may be needed in elderly patients with severe calcification of the ascending aorta.

Induction of isoflavonoid pathway in the model legume Lotus japonicus: molecular characterization of enzymes involved in phytoalexin biosynthesis.

Treatment of the seedlings of Lotus japonicus, a model legume for molecular genetic studies, with reduced glutathione (GSH) resulted in the accumulation of an isoflavan phytoalexin, vestitol. Using PCR strategies based on the conserved amino acid sequences, full length P450 cDNAs were obtained from GSH-treated seedling roots. When the clones, LjCYP-1 (CYP93C family) and LjCYP-2 (CYP81E family), were heterologously expressed in yeast, the proteins exhibited 2-hydroxyisoflavanone synthase (IFS) and isoflavone 2'-hydroxylase (I2'H) activities, respectively. The transcription levels of LjCYP-1, LjCYP-2 and isoflavone reductase, which are all involved in vestitol biosynthesis, coordinately increased upon elicitation. Genomic Southern blot analysis indicated that the IFS gene forms a small gene family and a single copy of the I2'H gene is present in the L. japonicus genome. Molecular biological aspects of P450s involved in the isoflavonoid pathway and the genomic approach to flavonoid metabolism in this unique plant are discussed.

Induction of isoflavonoid and retrochalcone branches of the flavonoid pathway in cultured Glycyrrhiza echinata cells treated with yeast extract.

Yeast extract-treated suspension cultures of a new cell line, AK-1, of Glycyrrhiza echinata were induced to produce an isoflavonoid phytoalexin (medicarpin) and metabolites of retrochalcone/flavone pathway (echinatin, licodione, and 7,4'-dihydroxyflavone). From these cells, putative full-length cDNAs encoding cytochrome P450s, (2S)-flavanone 2-hydroxylase and isoflavone 2'-hydroxylase, were cloned.

Cloning and functional expression of a cytochrome P450 cDNA encoding 2-hydroxyisoflavanone synthase involved in biosynthesis of the isoflavonoid skeleton in licorice.

Isoflavonoids are distributed predominantly in leguminous plants and play critical roles in plant physiology. A cytochrome P450 (P450), 2-hydroxyisoflavanone synthase, is the key enzyme in their biosynthesis. In cultured licorice (Glycyrrhiza echinata L., Fabaceae) cells, the production of both an isoflavonoid-derived phytoalexin (medicarpin) and a retrochalcone (echinatin) is rapidly induced upon elicitation. In this study, we obtained a full-length P450 cDNA, CYP Ge-8 (CYP93C2), from the cDNA library of elicited G. echinata cells. When the flavanones liquiritigenin and naringenin were incubated with the recombinant yeast microsome expressing CYP93C2, major products emerged and were readily converted to the isoflavones daidzein and genistein by acid treatment. The chemical structures of the products from liquiritigenin (2-hydroxyisoflavanone and isoflavone) were confirmed by mass spectrometry. CYP93C2 was thus shown to encode 2-hydroxyisoflavanone synthase, which catalyzes the hydroxylation associated with 1,2-aryl migration of flavanones. Northern-blot analysis revealed that transcripts of CYP93C2, in addition to those of other P450s involved in phenylpropanoid/flavonoid pathways, transiently accumulate upon elicitation.

Observation of Multi-TeV Gamma Rays from the Crab Nebula using the Tibet Air Shower Array.

The Tibet experiment, operating at Yangbajing (4300 m above sea level), is the lowest energy air shower array, and the new high-density array constructed in 1996 is sensitive to gamma-ray air showers at energies as low as 3 TeV. With this new array, the Crab Nebula was observed in multi-TeV gamma-rays and a signal was detected at the 5.5 sigma level. We also obtained the energy spectrum of gamma-rays in the energy region above 3 TeV which partially overlaps those observed with imaging atmospheric Cerenkov telescopes. The Crab spectrum observed in this energy region can be represented by the power-law fit dJ&parl0;E&parr0;&solm0;dE=&parl0;4.61+/-0.90&parr0;x10-12&parl0;E&solm0;3 TeV&parr0;-2.62+/-0.17 cm-2 s-1 TeV-1. This is the first observation of gamma-ray signals from point sources with a conventional air shower array using scintillation detectors.

Molecular cloning and biochemical characterization of a novel cytochrome P450, flavone synthase II, that catalyzes direct conversion of flavanones to flavones.

Cytochrome P450 cDNAs, AFNS2 and TFNS5, were isolated from snapdragon and torenia petal cDNA libraries, respectively, based on the sequence homology with licorice CYP93B1 cDNA encoding (2S)-flavanone 2-hydroxylase. They were expressed in yeast and identified to encode flavone synthase II catalyzing direct conversion of flavanones to flavones probably via 2-hydroxyflavanones.

CYP81E1, a cytochrome P450 cDNA of licorice (Glycyrrhiza echinata L.), encodes isoflavone 2'-hydroxylase.

The microsome of yeast cells overexpressing CYP81E1, a cytochrome P450 cDNA recently cloned from licorice (Glycyrrhiza echinata L., Fabaceae), catalyzed the hydroxylation of isoflavones, daidzein and formononetin, to yield 2'-hydroxyisoflavones, 2'-hydroxydaidzein, and 2'-hydroxyformononetin, respectively. The chemical structures of the reaction products were confirmed by mass spectrometric analysis. Genistein also yielded a putative 2'-hydroxylated product, but flavanones and cinnamic acid derivatives were not used as substrates for the reaction with the recombinant yeast microsome. CYP81E1 protein was thus demonstrated for the first time to be isoflavone 2'-hydroxylase involved in the biosynthesis of isoflavonoid-derived antimicrobial compounds of legumes.

Identification of a cytochrome P450 cDNA encoding (2S)-flavanone 2-hydroxylase of licorice (Glycyrrhiza echinata L.; Fabaceae) which represents licodione synthase and flavone synthase II.

The microsome of insect cells expressing CYP Ge-5 (CYP93B1), a cytochrome P450 cDNA of licorice (Glycyrrhiza echinata L.), catalyzed the formation of [14C]licodione and [14C]2-hydroxynaringenin from (2S)-[14C]liquiritigenin and (2S)-[14C]naringenin, respectively. On acid treatment, the products were converted to 14C-labeled 7,4'-dihydroxyflavone and apigenin. Eriodictyol was also converted to luteolin by the reaction with the microsome of yeast expressing CYP93B1 and subsequent acid treatment. CYP93B1 was thus shown to encode (2S)-flavanone 2-hydroxylase, which has previously been designated to licodione synthase and flavone synthase II depending on the substrates employed.

[Native aortic valve thrombus revealed by routine echocardiography: a case report].

A 75-year-old man presented with palpitations due to atrial flutter. Transthoracic echocardiography revealed a mobile aortic valve mass (17 mm in diameter) attached to the non-coronary cusp of the aortic valve. There was no evidence of hypercoagulative state. Computed tomography showed old cerebral infarction in the territory supplied by the right middle cerebral artery. The mass was surgically resected. The aortic valve was preserved because there were no organic changes in the valve. Histological examination demonstrated an organized thrombus. Only three cases of thrombus attached to the normal native aortic valve have been reported. Native aortic valve thrombus may be important in the differential diagnosis of aortic valve mass.

Anthocyanin-producing dandelion callus as a chalcone synthase source in recombinant polyketide reductase assay.

Purple-coloured dandelion (Taraxacum officinale) callus cultures producing anthocyanin pigments were established on a cytokinin-rich medium under the light. When the cells were placed in the dark, only grey cells proliferated. Anthocyanin productivity of these cells was partially restored in the light. The major pigment was identified as cyanidin 3-(6"-malonylglucoside). The lower stem of the original plant contained the same pigment. Chalcone synthase (CHS) activity was detected in the extracts of these purple cells, whereas no activity was observed in grey cells propagated in the dark. When the CHS-active cell-free extract was combined with the extract of Escherichia coli over expressing polyketide reductase (PKR) cDNA of licorice (Glycyrrhiza echinata), isoliquiritigenin (a 6'-deoxychalcone), in addition to naringenin (a 5-hydroxyflavanone), was detected as the reaction product from 4-coumaroyl-CoA, malonyl-CoA and NADPH. This result confirms the catalytic function of the PKR gene product.