RESUMEN
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.
Asunto(s)
Glycyrrhiza/enzimología , Plantas Medicinales , Animales , Células Cultivadas , Chalcona/análogos & derivados , Chalcona/metabolismo , Clonación Molecular , Sistema Enzimático del Citocromo P-450 , Glycyrrhiza/genética , Microsomas/metabolismo , Oxigenasas de Función Mixta , Datos de Secuencia Molecular , Resorcinoles/metabolismo , Saccharomyces cerevisiae , SpodopteraRESUMEN
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.
Asunto(s)
Aciltransferasas/metabolismo , Antocianinas/biosíntesis , Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Proteínas Bacterianas/metabolismo , Células Cultivadas , Escherichia coli/enzimología , Escherichia coli/metabolismo , Glycyrrhiza/enzimología , Extractos Vegetales/metabolismo , Extractos Vegetales/farmacología , Plantas/enzimología , Plantas MedicinalesRESUMEN
S-Adenosyl-L-methionine (SAM): isoliquiritigenin (2',4,4'-trihydroxychalcone) 2'-O-methyltransferase (CHMT) of alfalfa (Medicago sativa) catalyses the formation of 4,4'-dihydroxy-2'-methoxychalcone, which is the most potent inducer of nodulation-genes of Rhizobium meliloti, the symbiont of alfalfa which forms nitrogen-fixing nodules. SAM: licodione 2'-O-methyltransferase (LMT) is involved in the biosynthesis of a retrochalcone in cultured licorice (Glycyrrhiza echinata) cells and has been shown to be induced as a defence response of the cells. Because licodione exists in an equilibrium mixture of tautomeric 2',4,4',beta-tetrahydroxychalcone (major) and 1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)-1,3-propanedione (minor), the apparent mode of action of both enzymes is very similar. In this study, cultured alfalfa cells were shown to exhibit rapid and transient increases in the extractable activities of both CHMT and LMT after treatment with yeast extract (YE). Treatment of solution-cultured alfalfa seedlings with YE also resulted in a similar induction of both CHMT and LMT activities in the roots, but no activity was detected in the shoots. These activities were attributed to a single gene product, the CHMT protein, as extracts of Escherichia coli transformed with the CHMT cDNA exhibited both CHMT and LMT activities. In contrast, in G. echinata cells, LMT was induced after YE treatment, but no CHMT activity was observed. It is concluded that alfalfa CHMT and licorice LMT are distinct enzymes, the former displaying the wider substrate specificity.
Asunto(s)
Glycyrrhiza/enzimología , Medicago sativa/enzimología , Metiltransferasas/metabolismo , Plantas Medicinales , Chalcona/análogos & derivados , Chalcona/metabolismo , Chalconas , Isomerismo , Cinética , Resorcinoles/metabolismo , Especificidad por SustratoRESUMEN
A concerted international effort currently is being made to localize human genes and to identify their role in specific diseases. This will enable physicians to test for a broader range of genetic characteristics and to manipulate human somatic and germ-line cells. With this new age of genetics comes a host of ethical issues and questions.
Asunto(s)
Actitud , Asesoramiento Genético , Pruebas Genéticas , Femenino , Enfermedades Fetales/diagnóstico , Enfermedades Fetales/prevención & control , Enfermedades Fetales/terapia , Enfermedades Genéticas Congénitas/diagnóstico , Enfermedades Genéticas Congénitas/prevención & control , Enfermedades Genéticas Congénitas/terapia , Terapia Genética , Derechos Humanos , Humanos , Embarazo , Diagnóstico PrenatalRESUMEN
A 26-year-old woman was admitted to our hospital because of abdominal mass. She became ileus and an emergency operation was consequently performed. A histological examination of the abdominal tumor indicated proliferation of undifferentiated lymphoblasts with intermingled histiocytes so called "starry sky appearance". The diagnosis was Burkitt's lymphoma. On the 4th post-operative day, a cardiac tamponade became evident. The pericardial effusion contained many lymphoblasts and the diagnosis was pericarditis due to the invasion of lymphoma cells. The pericarditis was successfully treated by infusion of doxycycline into the pericardial space following drainage. The patient responded to systemic chemotherapy with complete remission. 7 courses of systemic chemotherapy along with intrathecal infusions for CNS prophylaxis were subsequently carried out. A state of complete remission has continued for more than 13 months. Cardiac tamponade accompanied by Burkitt's lymphoma is quite rare and has not ever been reported in Japan in our knowledge. The efficacy of surgical treatment before systemic chemotherapy and the series of intrathecal infusions for CNS prophylaxis was demonstrated in this case.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Linfoma de Burkitt/terapia , Taponamiento Cardíaco/cirugía , Adulto , Linfoma de Burkitt/complicaciones , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/cirugía , Taponamiento Cardíaco/etiología , Terapia Combinada , Ciclofosfamida/administración & dosificación , Citarabina/administración & dosificación , Doxorrubicina/administración & dosificación , Femenino , Humanos , Obstrucción Intestinal/etiología , Obstrucción Intestinal/cirugía , Metotrexato/administración & dosificación , Derrame Pericárdico/tratamiento farmacológico , Derrame Pericárdico/cirugía , Derrame Pleural/tratamiento farmacológico , Derrame Pleural/cirugía , Inducción de Remisión , Vincristina/administración & dosificaciónRESUMEN
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.
Asunto(s)
Dinoprostona/fisiología , Fibroblastos/fisiología , Hígado/citología , Receptores de Prostaglandina E/agonistas , Receptores de Prostaglandina/agonistas , Animales , Calcio/metabolismo , Tamaño de la Célula , Supervivencia Celular , Células Cultivadas , Dinoprostona/farmacología , Activación Enzimática , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Masculino , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Prostaglandina/biosíntesis , Receptores de Prostaglandina E/biosíntesis , Subtipo EP3 de Receptores de Prostaglandina ERESUMEN
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.
RESUMEN
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.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Glycyrrhiza/enzimología , Glycyrrhiza/genética , Oxigenasas de Función Mixta/genética , Plantas Medicinales , Clonación Molecular , Genes de Plantas/genética , Isoflavonas/metabolismo , Modelos Moleculares , Datos de Secuencia MolecularRESUMEN
The crude extract prepared from Glycyrrhiza echinata cells treated with yeast extract catalyzed the formation of liquiritigenin (5-deoxyflavanone) and isoliquiritigenin (6'-deoxychalcone) in addition to naringenin (5-hydroxyflavanone) when incubated with 4-coumaroyl-CoA and malonyl-CoA in the presence of high concentrations (0.1 mM or higher) of NADPH. Incubation without NADPH, or with low concentrations (0.01 mM or lower), gave only naringenin as a reaction product. With NADH (1 mM), the major product was naringenin accompanied by a small quantity of liquiritigenin. The initial product of the assay with 1 mM NADPH was isoliquiritigenin, indicating a reaction catalyzed by 6'-deoxychalcone synthase (DOCS). Subsequent formation of liquiritigenin was attributed to the presence of chalcone isomerase in the crude extract. The results constitute the first demonstration in vitro of DOCS activity which, in G. echinata cells and other leguminous plants, is involved in the biosynthesis of retrochalcone and 5-deoxyisoflavonoid-derived phytoalexins.
Asunto(s)
Aciltransferasas/biosíntesis , Glycyrrhiza/enzimología , Plantas Medicinales , Sistema Libre de Células , Inducción Enzimática/efectos de los fármacos , Alcaloides de Pirrolicidina/biosíntesis , Levaduras/análisisRESUMEN
Chalcone synthase activity catalyzing the formation of naringenin (5-hydroxyflavanone) was detected in cell suspension cultures of Glycyrrhiza echinata. This activity rapidly increased by treatment of the cells with yeast extract, while non-treated cells showed a constant low activity. Isolated G. echinata protoplasts accumulated retrochalcone (echinatin) and its biosynthetic intermediate (licodione) during 24 h of culture. When the protoplasts were incubated with [(14)C(U)]phenylalanine, liquiritigenin (5-deoxyflavanone) was transiently labeled, indicating the induction of 6'-deoxychalcone synthase. The formation of liquiritigenin, in addition to naringenin, was observed when the crude extracts from the protoplasts were assaved for CHS activity.
RESUMEN
Transfer into a fresh medium or immobilization by entrapment in calcium alginate gels of cultured Glycyrrhiza echinata cells caused a rapid and transient accumulation of a retrochalcone, echinatin, in both the cells and the medium. The higher level and longer duration of echinatin production was observed in the immobilized cells than in freely suspended cells. Transfer of the cells into the medium containing either sodium alginate or calcium chloride, and the addition of sodium alginate into the suspension culture, caused the same effect as observed in cell immobilization. A novel metabolite was also detected in the induced cells. Activities of the enzymes involved in echinatin biosynthesis were shown to rapidly increase by immobilization of the cells.
RESUMEN
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.
Asunto(s)
Chalcona/metabolismo , Flavonoides/biosíntesis , Glycyrrhiza/metabolismo , Liasas Intramoleculares/metabolismo , Isoenzimas/metabolismo , Células Cultivadas , Glycyrrhiza/citología , Glycyrrhiza/enzimología , Especificidad por SustratoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Isoflavonas/química , Isoflavonas/metabolismo , Sistema Libre de Células , Cromatografía Líquida de Alta PresiónRESUMEN
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.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Glycyrrhiza/enzimología , Glycyrrhiza/genética , Isoflavonas/metabolismo , Plantas Medicinales , Secuencia de Aminoácidos , Línea Celular , Clonación Molecular , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , ADN Complementario , Biblioteca de Genes , Cinética , Microsomas/enzimología , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Three O-methyltransferases which catalyze S-adenosyl-L-methionine (SAM)-dependent O-methylation of licodione (LMT), flavone/flavonol (FMT), and caffeic acid (CMT) were separated from the callus culture of Glycyrrhiza echinata, and characteristic differences between their pH optima and Mg(2+) requirement for activity were demonstrated. The activity of LMT, which is involved in retrochalcone (echinatin) biosynthesis, but not of FMT or CMT, was found to be stimulated when suspension-cultured G. echinata cells were treated with yeast extract (YE), which causes rapid production of echinatin in the cells. Cycloheximide suppressed both the YE-induced echinatin formation and LMT enhancement. The results indicate a selective induction of retrochalcone pathway in Glycyrrhiza cells in response to stress.
RESUMEN
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.
Asunto(s)
Fibroma/patología , Neoplasias Cardíacas/patología , Ecocardiografía , Femenino , Fibroma/diagnóstico por imagen , Neoplasias Cardíacas/diagnóstico por imagen , Humanos , Persona de Mediana EdadRESUMEN
The incorporation of [1-(14)C]acetate and [2(14) C]mevalonate into free and esterified triterpen-3-ols was examined in original plant organs and tissue cultures of Glycyrrhiza glabra var. glandulifera. Both substrates labeled ß-amyrin, an oleanane-type triterpene, and cycloartenol and 24-methylenecycloartanol, both of which are intermediates of phytosterol biosynthesis. The label in esterified triterpenes was distributed mainly in phytosterol intermediates, but not in ß-amyrin. The ratio of ßamyrin formation among the three triterpenes from [2-(14)C]mevalonate was relatively high in stolon segments and in root cultures, but negligible in callus cultures. Administration of a specific inhibitor of squalene-2, 3-epoxide:cycloartenol (lanosterol) cyclase caused a marked increase of ß-amyrin synthesis in root suspension cultures, and of 24-methylenecycloartanol synthesis in cell suspension cultures, from [2-(14)C]mevalonate.
RESUMEN
Cultured Papaver bracteatum cells accumulate large amounts of dopamine (0.1-4 mg/g fresh wt.) and much lower levels of the alkaloids, thebaine (0-6 µg/g fresh wt.) and sanguinarine (10-500 µg/g fresh wt.). Upon removal of hormone from their culture media, organogenesis and an elevation in morphinan alkaloid concentration were observed. Light and electron microscopic studies reveal that a correlation exists between the appearance of laticifer-like cells and thebaine in these cells.